Nematode neuromuscular junction GABA receptors and methods related thereto

ABSTRACT

The present invention relates to the field of GABA receptor structure and function. In one aspect, the present invention relates to impairment of the gamma-aminobutyric acid (GABA) receptors in parasitic nematodes, for the purpose of crop protection and/or soil treatment. The invention includes mutated nematode GABA receptor subunits, nematode GABA neuromuscular junction receptor complexes, nucleic acids which encode the mutated and functional receptor complexes, antibodies which selectively bind the GABA receptor complexes and/or subunits, and assays for compounds which adversely affect nematode GABA neuromuscular junction receptor function. The present invention therefore relates broadly to recombinant DNA technology, molecular biology tools, and crop protection and/or soil treatment.

[0001] The present invention claims priority to U.S. Provisional PatentApplication Serial No. 60/107,727, filed Nov. 9, 1998.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of GABA receptorstructure and function. In one aspect, the present invention relates toimpairment of the gamma-aminobutyric acid (GABA) receptors in parasiticnematodes, for the purpose of crop protection and/or soil treatment. Theinvention includes mutated nematode GABA receptor subunits, nematodeGABA neuromuscular junction receptor complexes, nucleic acids whichencode the mutated and functional receptor complexes, antibodies whichselectively bind the GABA receptor complexes and/or subunits, and assaysfor compounds which adversely affect nematode GABA neuromuscularjunction receptor function. The present invention therefore relatesbroadly to recombinant DNA technology, molecular biology tools, and cropprotection and/or soil treatment.

[0003] The present invention also relates to the fields of GABAneurotransmitter research, particularly with regard to drug discovery.In that regard, the invention includes assays for substances whichaffect GABA receptors, as well as the tools necessary to conduct suchassays, such as nucleic acids, amino acids, antibodies, vectors and celllines.

BACKGROUND OF THE INVENTION

[0004] Nematodes (nema—thread; oides—resembling), which are unsegmentedroundworms with elongated, fusiform, or saclike bodies covered withcuticle, are virtually ubiquitous in nature, inhabiting soil, water andplants, and are importantly involved in a wide range of animal and plantparasitic diseases.

[0005] Nematodes are Animal Pathogens

[0006] The roundworm parasites of mammals belong to the phylumNemathelminthes. The roundworms include the hookworm (e.g. Necatoramericanus and Ancylostoma duodenale), roundworm (e.g. the commonroundworm Ascaris lumbricoides), whipworm (e.g. Trichuris trichiura),and the pinworm or threadworm (e.g. Enterobius vermicularus). as well asStrongyloides stercoralis, Trichinella spiralis and the filarial wormWuchereria bancrofti. Other important roundworm parasites includeAncylostoma caninum (infections of man), Strongylus vulgaris (infectionsof horses), Trichostrongylus colubriformis, Ostertagia circumcincta(infections of sheep and goats), Haemonchus contortus (infections ofsheep and goats), Ostertagia ostertagi, Haemonchus placei (infections ofcattle), Ascaris suum (infections of pigs), Toxascaris leonina orUncinaria stenocephala (infections of dogs), Toxocara spp (circulatoryinfections of man) and Dirofilaria immitis (circulatory infections ofcats and dogs).

[0007] Even when symptom-free, parasitic worm infections are harmful tothe host animal for a number of reasons; e.g. they deprive the host offood, injure organs or obstruct ducts, may elaborate substances toxic tothe host, and provide a port of entry for other organisms. In othercases, the host may be a species raised for food and the parasite may betransmitted upon eating to infect the ingesting animal. It is highlydesirable to eliminate such parasites as soon as they have beendiscovered.

[0008] More commonly, such infections are not symptom-free. Helminthinfections of mammals, particularly by parasitic nematodes, are a sourceof great economic loss, especially of livestock and pets, e.g. sheep,cattle, horses, pigs, goats, dogs, cats, and birds, especially poultry(see CSIRO/BAE Report—“Socio-economic Developments and Trends in theAgricultural Sector: Implications for Future Research”). These animalsmust be regularly treated with anthelminthic chemicals in order to keepsuch infections under control, or else the disease may result inanaemia, diarrhoea, dehydration, loss of appetite, and even death.

[0009] The only currently available means for controlling helminthinfections is with the use of anthelminthic chemicals, but these areonly effective against resident worms present at the time of treatment.Therefore, treatment must be continuous since the animals are constantlyexposed to infection; e.g. anthelminthic treatment withdiethylcarbamazine is required every day or every other day most of theyear to control Dirofilaria immitis or the dog heartworm. This is anexpensive and labour intensive procedure. Due to the widespread use ofanthelminthic chemicals, the worms may develop resistance and so new andmore potent classes of chemicals must be developed. An alternativeapproach is clearly desirable.

[0010] The accepted methodology for pharmaceutical control of nematodeshas centered around the use of the drug benzimidazole and its congeners.The use of these drugs on a wide scale has led to many instances ofresistance among nematode populations (Prichard et al., 1980; Coles,1986). There are more than 100,000 described species of nematodes.

[0011] Other options for a drug treatment include employing anavermectin, pyrantel, morantel, closantel, praziquantel etc.Benzimidazole(s) or benzimidazole prodrug treatments includeoxfendazole, thiabendazole, albendazole, cambenazole, fenbendazole,flubendazole, mebendazole, oxibendazole, parbendazole, thiophanate,febantel and netobimin.

[0012] Nematodes are Plant Pathogens

[0013] Nematodes thrive in virtually all environments throughout theworld and are one of the largest and most diverse groups ofmulticellular organisms. Many species are parasites of agronomic crops,but other species are beneficial to agriculture. Nematodes thatparasitize plants cause an estimated $8 billion annual loss (12%) toU.S. growers and nearly $78 billion loss globally. For example, thesoybean cyst nematode causes annual losses in the North Central Regionof the U.S. amounting to $267 million.

[0014] Traditional nematode reduction methods usually rely on acombination of petroleum byproduct soil treatments and crop rotation.Stricter environmental regulations are forcasted to limit the use ofpetroleum by-products, and threaten to impact agriculture if no saferalternatives are found or invented. A review of the imact of nematodeson crops can be found in: Hussey, R. Plant & Soil Nematodes: SocietalImpact and Focus for the Future and can be obtained by writing toDepartment of Plant Pathology, University of Georgia, Athens, Ga. USA30602-7274.

[0015] GABA Receptors

[0016] The gamma-aminobutyric acid receptors (GABA receptors) are themost abundant inhibitory receptor in the mammalian brain. They arecomprised of a heteropolymeric structure that form a chloride ionchannel, and contain multiple recognition sites for the binding ofmolecules. The binding of GABA to its specific recognition site on the aGABA receptor opens the ion channel and allows chloride ions to flowinto the nerve cell. This action hyperpolarizes the cell membrane ofthat neuron and thereby makes the cell less reactive to excitatorystimuli. The chloride ion current may also be regulated by various drugsthat serve as positive or negative modulators of the GABA receptor(Puia, G. et al. Molecular Pharm. 1991, 39, 691).

[0017] Many clinical conditions are thought to arise, in part, from theimbalance between neurotransmission of GABA and those of otherneurotransmitters. These conditions include Huntington's chorea,Parkinson's disease, spasticity, epilepsy, schizophrenia and tardivedyskinesia. Decreased GABA activity appears to contribute to thepathogenesis of these diseases. In addition, analgesia and satiety arethought to be regulated by GABA activity. Methods of modifying GABAergicneurotransmission are therefore desirable in order to modify theseconditions.

[0018] The so-called benzodiazepine (BZD) receptor is a site for suchallosteric modulators on one class of the GABA receptor, the GABA-Areceptor. This site mediates two opposing effects, one that amplifiesthe action of GABA (“positive” efficacy) and the other that reduces theaction of GABA (“negative” efficacy). Agents facilitatingGABA-receptor/chloride ion-channel functions via the BZD site arereferred to as agonists, while agents reducing such function arereferred to as inverse agonists. Antagonists at this site block theeffects of agonists or inverse agonists by competitively inhibitingtheir binding. It is thus possible to have a series of compounds inwhich members equally bind to the BZD site but have equal and oppositeregulatory effects on the GABA-A receptor/chloride ion channel. Also,within the series a continuum of activity is possible (Takada, S. et al.J. Med. Chem. 1988, 31, 1738). Thus, BZD receptor ligands can induce awide spectrum of pharmacological effects ranging from muscle relaxant,hypnotic, sedative, anxiolytic, and anticonvulsant activities, producedby full or partial agonists (“positive”), to the proconvulsant,anti-inebriant, and anxiogenic activities, produced by inverse agonists(“negative”). (A further understanding of this area can be gleaned from:Mohler, H. Arzneim.-Forsch./Drug Res.1992, 42 (2a), 211; Haefely, W. etal., Advances in Drug Research, Academic Press, vol. 14, 1985, pp.165-322; Skolnick, P. et al., GABA and Benzodiazepine Receptors,Squires, R., Ed., 1987, pp. 99-102 and references cited therein.)

[0019] The fourth edition of the Diagnostic and Statistical Manual ofMental Disorders (DSM-IV.TM.), published in 1994 by the AmericanPsychiatric Association, Washington, D.C., defines anxiety and relateddisorders. These are panic disorder without agoraphobia, panic disorderwith agoraphobia, agoraphobia without history of panic disorder,specific phobia, social phobia, obsessive-compulsive disorder,post-traumatic stress disorder, acute stress disorder, generalizedanxiety disorder, anxiety disorder due to a general medical condition,substance-induced anxiety disorder and anxiety disorder not otherwisespecified.

[0020] Anxiety disorders are generally treated by counseling or withdrugs. Classes of drugs which are widely prescribed for the treatment ofanxiety disorders include the benzodiazepines (such as diazepam) andbuspirone hydrochloride.

[0021] Several animal models have been developed which are recognized inthe art as being predictive of anxiolytic activity. These include thefear-potentiated startle model, described by Davis in Psychopharmacology62:1; 1979, Behav. Neurosci. 100:814;1986 and TiPS, January 1992 Vol.13, 35-41, the elevated plus model described by Lister inPsychopharmacol. 92:180-185; 1987, and the well-knownpunished—responding (conflict) model, described, for example, in“Psychopharmacology of Anxiolytics and Antidepressants”, edited by S. E.File, pp. 131-153, Raven Press, New York, 1991.

[0022] Citation of the above documents is not intended as an admissionthat any of the foregoing is prior art. All statements as to the date orrepresentation as to the contents of these documents is based onsubjective characterization of information available to the applicant,and does not constitute any admission as to the accuracy of the dates orcontents of the documents.

SUMMARY OF THE INVENTION

[0023] The present invention therefore provides nematode neuromuscularjuntion GABA receptor complexes of the formula I

A-B  Formula I

[0024] wherein A an amino acid sequence selected from the groupconsisting of:

[0025] (a) an amino acid sequence which is encoded by a nucleic acidsequence which has at least 80% identity to SEQ ID NO 2; wherein saididentity can be determined using the DNAsis computer program and defaultparameters; and

[0026] (b) an amino acid sequence which has at least 80% identity to SEQID NO 1; wherein said identity can be determined using the DNAsiscomputer program and default parameters; and

[0027] wherein B is an amino acid sequence selected from the groupconsisting of:

[0028] (a) an amino acid sequence which is encoded by a nucleic acidsequence which has at least 80% identity to SEQ ID NO 4; wherein saididentity can be determined using the DNAsis computer program and defaultparameters; and

[0029] (b) an amino acid sequence which has at least 80% identity to SEQID NO 3; wherein said identity can be determined using the DNAsiscomputer program and default parameters; and

[0030] wherein A-B is a heteropentamer which comprises 1 to 4 A aminoacid sequences and 1 to 4 B amino acid sequences.

[0031] Also provided are homopentamer nematode neuromuscular juntionGABA receptor complexes of the formula II

B₅  Formula II

[0032] wherein B is an amino acid sequence selected from the groupconsisting of:

[0033] (a) an amino acid sequence which is encoded by a nucleic acidsequence which has at least 80% identity to SEQ ID NO 4; wherein saididentity can be determined using the DNAsis computer program and defaultparameters; and

[0034] (b) an amino acid sequence which has at least 80% identity to SEQID NO 3; wherein said identity can be determined using the DNAsiscomputer program and default parameters.

[0035] Also provided are nematode neuromuscular junction GABA receptorcomplex subunits, comprising an amino acid sequence selected from thegroup consisting of:

[0036] (a) an amino acid sequence which is encoded by a nucleic acidsequence which has at least 80% identity to SEQ ID NO 2; wherein saididentity can be determined using the DNAsis computer program and defaultparameters; and

[0037] (b) an amino acid sequence which has at least 80% identity to SEQID NO 1; wherein said identity can be determined using the DNAsiscomputer program and default parameters.

[0038] Also provided are nematode neuromuscular junction GABA receptorcomplex subunits, comprising an amino acid sequence selected from thegroup consisting of:

[0039] (a) an amino acid sequence which is encoded by a nucleic acidsequence which has at least 80% identity to SEQ ID NO 4; wherein saididentity can be determined using the DNAsis computer program and defaultparameters; and

[0040] (b) an amino acid sequence which has at least 80% identity to SEQID NO 3; wherein said identity can be determined using the DNAsiscomputer program and default parameters.

[0041] Also provided are nucleic acid compound which encodes a nematodeneuromuscular junction GABA receptor complex subunits, comprising:

[0042] (a) a nucleic acid sequence which has at least 80% identity toSEQ ID NO 2; wherein said identity can be determined using the DNAsiscomputer program and default parameters;

[0043] (b) a nucleic acid which encodes an amino acid sequence which hasat least 80% identity to SEQ ID NO 1; wherein said identity can bedetermined using the DNAsis computer program and default parameters;

[0044] (c) a nucleic acid sequence which is an allelic variant of SEQ IDNO 2; and

[0045] (d) a nucleic acid sequence fully complementary to a nucleic acidsequence selected from the group consisting of: a nucleic acid sequenceof (a); a nucleic acid sequence of (b); and a nucleic acid sequence of(c).

[0046] Also provided are nucleic acid compounds which encode a nematodeneuromuscular junction GABA receptor complex subunit, comprising anucleic acid sequence selected from the group consisting of:

[0047] (a) a nucleic acid sequence which has at least 80% identity toSEQ ID NO 4; wherein said identity can be determined using the DNAsiscomputer program and default parameters;

[0048] (b) a nucleic acid which encodes an amino acid sequence which hasat least 80% identity to SEQ ID NO 3; wherein said identity can bedetermined using the DNAsis computer program and default parameters;

[0049] (c) a nucleic acid sequence which is an allelic variant of SEQ IDNO 4; and

[0050] (d) a nucleic acid sequence fully complementary to a nucleic acidsequence selected from the group consisting of: a nucleic acid sequenceof (a); a nucleic acid sequence of (b); and a nucleic acid sequence of(c).

[0051] Also provided are nucleic acid compounds which encode a nematodeneuromuscular junction GABA receptor complex, comprising a first andsecond nucleic acid sequence, wherein said first nucleic acid sequenceis selected from the group consisting of:

[0052] (a) a nucleic acid sequence which has at least 80% identity toSEQ ID NO 2; wherein said identity can be determined using the DNAsiscomputer program and default parameters;

[0053] (b) a nucleic acid which encodes an amino acid sequence which hasat least 80% identity to SEQ ID NO 1; wherein said identity can bedetermined using the DNAsis computer program and default parameters;

[0054] (c) a nucleic acid sequence which is an allelic variant of SEQ IDNO 2; and

[0055] (d) a nucleic acid sequence which has at least 80% identity toSEQ ID NO 4; wherein said identity can be determined using the DNAsiscomputer program and default parameters;

[0056] (e) a nucleic acid which encodes an amino acid sequence which hasat least 80% identity to SEQ ID NO 3; wherein said identity can bedetermined using the DNAsis computer program and default parameters;

[0057] (f) a nucleic acid sequence which is an allelic variant of SEQ IDNO 4; and

[0058] wherein said second nucleic acid sequence is selected from thegroup consisting of:

[0059] (a) a nucleic acid sequence which has at least 80% identity toSEQ ID NO 4; wherein said identity can be determined using the DNAsiscomputer program and default parameters;

[0060] (b) a nucleic acid which encodes an amino acid sequence which hasat least 80% identity to SEQ ID NO 3; wherein said identity can bedetermined using the DNAsis computer program and default parameters;

[0061] (c) a nucleic acid sequence which is an allelic variant of SEQ IDNO 4.

[0062] Moreover, there are provided isolated antibodies selective for aGABA receptor complexes of the present invention.

[0063] In addition, there are provided methods to determine a testsubstance's ability to interact with a nematode neuromuscular junctionGABA receptor complex, comprising contacting a receptor complex of thepresent invention with a test substance and determining whether saidtest substance and said receptor complex interact.

[0064] Other methods include those to detect GABA receptors in a testsample, comprising: (a) immobilizing a test sample on a substrate; (b)contacting the test sample with an antibody of the present inventionunder conditions suitable for formation of a GABA receptor:antibodycomplex bound to the substrate; (c) removing non-bound material from thesubstrate under conditions that retain GABA:antibody complex binding tothe substrate; and (d) detecting the presence of the GABAreceptor:antibody complex.

[0065] Preferred isolated nucleic acid compounds of the presentinvention are those which comprise a nucleic acid sequence selected fromthe group consisting of: SEQ ID NO 2; SEQ ID NO 4; SEQ ID NO 6; SEQ IDNO 8; SEQ ID NO 10; and SEQ ID NO 12.

[0066] Preferred isolated amino acid compound of the present inventionare those which comprise a nucleic acid sequence selected from the groupconsisting of: SEQ ID NO 1; SEQ ID NO 3, SEQ ID NO 5; SEQ ID NO 7; SEQID NO 9; SEQ ID NO 11.

[0067] Moreover, for the purposes of the present invention, the term “a”or “an” entity refers to one or more of that entity; for example, “aprotein” or “a nucleic acid molecule” refers to one or more of thosecompounds or at least one compound. As such, the terms “a” (or “an”),“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprising”, “including”, and“having” can be used interchangeably. Furthermore, a compound “selectedfrom the group consisting of” refers to one or more of the compounds inthe list that follows, including mixtures (i.e., combinations) of two ormore of the compounds. According to the present invention, an isolated,or biologically pure, protein or nucleic acid molecule is a compoundthat has been removed from its natural milieu. As such, “isolated” and“biologically pure” do not necessarily reflect the extent to which thecompound has been purified. An isolated compound of the presentinvention can be obtained from its natural source, can be produced usingmolecular biology techniques or can be produced by chemical synthesis.

BRIEF DESCRIPTION OF THE FIGURES

[0068]FIG. 1. Cloning and genomic structure of unc-49 The genetic andphysical map in the unc-49 region of chromosome III, and the structureof cosmid T21C12.

[0069]FIG. 2. unc-49 produces three distinct GABA receptor subunits A)Structure of the unc-49 locus showing the positions of conserved GABAreceptor structural motifs. Domain structure of the locus is indicatedby bars at top (see text). B) unc-49 mRNA structure. Transcripts wereisolated both from cDNA libraries and from RT-PCR experiments. The shortarrows and circled numbers represent PCR primers. Two superimposedprimers (for example 68 and 73) represent a set of nested PCR primers.The shaded boxes represent coding exons and the open boxes representuntranslated regions. The SL1 splice leader was found at the 5′ ends ofthe mRNA species where indicated. C) Northern analysis. The probes,indicated below each lane, correspond to the carboxy-terminal repeats.Labels to the right of each lane indicate the probable identity of eachband. In the UNC-49C lane, the UNC-49B mRNA is visible because itcontains the UNC-49C open reading frame in its 3′ UTR. Asterisksindicate higher molecular weight bands which may correspond topartially-spliced unc-49 pre-mRNA. All lanes were exposed for the samelength of time.

[0070]FIG. 3. Structural overlap among unc-49 subunits UNC-49A, UNC-49Band UNC-49C are identical over the amino-terminal 40% of their length,but contain different putative GABA binding domains and transmembranedomains. Left panel shows an alignment of each subunit mRNA (bar at topindicates origin of exons encoding each portion). Triangle indicates theposition of the alternative splice site in UNC-49B. Note that theUNC49Cshort subunit is identical to the unique carboxy-terminal portionof UNC-49C, but lacks the entire amino terminus common to the othersubunits; in its place are four unique N-terminal amino acids (graybox). Right panel depicts the predicted unc-49 subunit proteins. Colorsindicate the genomic origin of the exons encoding each segment (see FIG.2).

[0071]FIG. 4. GABA receptor family A) Dendrogram of GABA receptorsubunits. The three unc-49 subunits do not correspond to any of thevertebrate classes of GABA_(A) receptor subunits. Alignments wereperformed using the Pileup program in the Genetics Computer Groupanalysis package. B) Sequence alignment of unc-49 subunits. Residues inblack boxes are conserved in all members of a set of sevenrepresentative non-C. elegans GABA receptor subunits, and residues ingray boxes are conserved in six out of seven members of this set (seeExperimental Procedures). The rat β2 GABA_(A) and Drosophila RDLreceptor subunits are included for comparison. Residues conserved in allmembers of the ligand-gated ion channel superfamily are indicated byasterisks. Dashed line indicates the disulfide bonded loop motif (CX₁₃C)conserved in all ligand-gated ion channel subunits. Bars labeled BDI andBDII indicate putative GABA binding domains and bars labeled M1-M4indicate membrane-spanning domains. Residues in BDI and BDII which arefunctionally important in the ρ and β GABA receptor subunits, but aredivergent in the C. elegans subunits, are denoted by # and $,respectively. Glutamic acid residue in UNC-49C M2 is denoted by @.Arrowheads indicate predicted sites of signal peptide cleavage forUNC-49B and UNC-49C and the rat β2 subunit. Residues are numbered fromthe predicted start of translation except for the rat β2 subunit, whichis numbered from the predicted signal peptide cleavage site according toconvention. UNC-49B is numbered according to the UNC-49B.1 sequence C)Residues comprising the M3-M4 intracellular loops of the unc-49-encodedsubunits. Sequences of the three UNC-49B isoforms are also shown.Intracellular loop sequences have not been aligned. Symbols above eachintracellular loop indicate potential regulatory phosphorylation sites(A indicates a PKA site, C indicates a PKC site, and * indicates a CKIIsite).

[0072]FIG. 5. All unc-49 mutations affect UNC-49B A) Upper panel,Southern blot of EcoRV-digested genomic DNA probed with T21C12 insertDNA. Numbers at right indicate the positions of DNA size standards.Lower panel, a restriction map of the genomic DNA encompassing unc-49.Positions of the polymorphisms observed in unc-49(n1324) andunc-49(e929) are shown beneath the restriction map. Shaded bar indicatesthe T21C12.1 open reading frame. B) Positions of mutations in the unc-49alleles are shown. e382, e468, e641, and e929 affect only UNC-49Bwhereas e407, n1324, and n2392 affect UNC-49A, UNC-49B and UNC-49C. Barsat top represent unc-49 domains. C) Summary of unc-49 mutations.

[0073]FIG. 6. UNC-49B and UNC-49C are co-localized A) Structure ofUNC-49::GFP transgenes. The left panel shows the site at which GFP wasinserted, in frame, into the unc-49 rescuing fragment. Vertical barsrepresent transmembrane domains. Right panel shows the subunits whichare produced by the transgene. ‘GFP’ indicates subunits tagged withGFP, + indicates wild-type subunits, − indicates inactivated subunits.B, C) Confocal images of UNC-49B::GFP and UNC-49C::GFP transgenic worms.In the head (left panels), both constructs produce GFP fluorescence inthe nerve ring (nr) and head muscle arms (ma). In the vicinity of thevulva (vul, middle panels), both constructs produce bright fluorescencein the dorsal nerve cord (dc). Fluorescence in the ventral nerve cordwas not clearly visible in these specimens because of their orientationon the microscope slide. Bright spots in the middle of the animal areautofluorescent gut granules. In the tail, the UNC-49B::GFP constructproduces fluorescence in the sphincter muscle (sph) while theUNC-49C::GFP construct does not (right panels). This individual showedsome GFP fluorescence in the body wall muscles in the tail (bwm).

[0074]FIG. 7. UNC-49B and UNC-49C associate at the neuromuscularjunction. A) Structure of the modified UNC-49::GFP transgenes. Leftpanel shows transgene structure and right panel shows the subunitsproduced by each transgene, as in FIG. 6. B) Epifluorescent image ofUNC-49B::GFP expressed in the absence of UNC-49C. Fluorescence wasvisible along the ventral nerve cord (vc), suggesting proper synapticlocalization of UNC-49B::GFP. C) UNC-49C::GFP expressed in the absenceof UNC-49B. Muscle membranes and muscle arms (ma) are fluorescent, butno enrichment of fluorescence is observed in the ventral nerve cord (vc)indicating that UNC-49C::GFP is not synaptically localized.

[0075]FIG. 8. UNC-49B and UNC-49C co-assemble in heterologous cells A)Response of a representative UNC-49B.1-injected oocyte to 10 sec pulsesof GABA at 10 μM, 30 μM, 60 μM and 100 μM. B) GABA dose response curvesobtained from Xenopus oocytes injected with UNC-49B (circles) orUNC-49B+UNC-49C (squares). Error bars represent standard error of themean. C) Single-channel recordings from HEK-293 cells expressing UNC-49Balone (top trace) or UNC-49B+UNC-49C (bottom trace).

[0076]FIG. 9. Proposed interaction of UNC-49B and UNC-49C at thesynapse. UNC-49B and UNC-49C form a heteromeric GABA receptor which istethered at the synapse by the binding of UNC-49B with a cytoskeletalcomponent of the postsynaptic specialization.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0077] SEQ ID NO Sequence Description 1 unc 49-C amino acid sequence 2unc 49-C DNA sequence 3 une 49-B amino acid sequence 4 unc 49-B DNAsequence 5 unc 49-A amino acid sequence 6 unc 49-A DNA sequence 7 unc49-B2 amino acid sequence 8 unc 49-B2 DNA sequence 9 unc 49-B3 aminoacid sequence 10 unc 49-B3 DNA sequence 11 unc 49-Cshort amino acidsequence 12 unc 49-Cshort DNA sequence

DETAILED DESCRIPTION OF THE INVENTION

[0078] The present invention therefore provides nematode neuromuscularjuntion GABA receptor complexes of the formula I

A-B  Formula I

[0079] wherein A an amino acid sequence selected from the groupconsisting of:

[0080] (a) an amino acid sequence which is encoded by a nucleic acidsequence which has at least 80% identity to SEQ ID NO 2; wherein saididentity can be determined using the DNAsis computer program and defaultparameters; and

[0081] (b) an amino acid sequence which has at least 80% identity to SEQID NO 1; wherein said identity can be determined using the DNAsiscomputer program and default parameters; and

[0082] wherein B is an amino acid sequence selected from the groupconsisting of:

[0083] (a) an amino acid sequence which is encoded by a nucleic acidsequence which has at least 80% identity to SEQ ID NO 4; wherein saididentity can be determined using the DNAsis computer program and defaultparameters; and

[0084] (b) an amino acid sequence which has at least 80% identity to SEQID NO 3; wherein said identity can be determined using the DNAsiscomputer program and default parameters; and

[0085] wherein A-B is a heteropentamer which comprises 1 to 4 A aminoacid sequences and 1 to 4 B amino acid sequences. For instance, A-B maycomprise 4 A amino acid sequences, and 1 B amino acid sequence, or A-Bmay comprise 3 A amino acid sequences, and 2 B amino acid sequences, orA-B may comprise 2 A amino acid sequences, and 3 B amino acid sequences,or A-B may comprise 1 A amino acid sequence, and 4 B amino acidsequences. So long as the amino acid sequences form a heteropentamerreceptor complex which, given the proper conditions, will allow chlorideion conductance, the complex is within the scope of the presentinvention.

[0086] Also provided are homopentamer nematode neuromuscular juntionGABA receptor complexes of the formula II

B₅  Formula II

[0087] wherein B is an amino acid sequence selected from the groupconsisting of:

[0088] (a) an amino acid sequence which is encoded by a nucleic acidsequence which has at least 80% identity to SEQ ID NO 4; wherein saididentity can be determined using the DNAsis computer program and defaultparameters; and

[0089] (b) an amino acid sequence which has at least 80% identity to SEQID NO 3; wherein said identity can be determined using the DNAsiscomputer program and default parameters.

[0090] Also provided are nematode neuromuscular junction GABA receptorcomplex subunits, comprising an amino acid sequence selected from thegroup consisting of:

[0091] (a) an amino acid sequence which is encoded by a nucleic acidsequence which has at least 80% identity to SEQ ID NO 2; wherein saididentity can be determined using the DNAsis computer program and defaultparameters; and

[0092] (b) an amino acid sequence which has at least 80% identity to SEQID NO 1; wherein said identity can be determined using the DNAsiscomputer program and default parameters.

[0093] Also provided are nematode neuromuscular junction GABA receptorcomplex subunits, comprising an amino acid sequence selected from thegroup consisting of:

[0094] (a) an amino acid sequence which is encoded by a nucleic acidsequence which has at least 80% identity to SEQ ID NO 4; wherein saididentity can be determined using the DNAsis computer program and defaultparameters; and

[0095] (b) an amino acid sequence which has at least 80% identity to SEQID NO 3; wherein said identity can be determined using the DNAsiscomputer program and default parameters.

[0096] Preferred isolated amino acid compound of the present inventionare those which comprise a nucleic acid sequence selected from the groupconsisting of: SEQ ID NO 1; SEQ ID NO 3, SEQ ID NO 5; SEQ ID NO 7; SEQID NO 9; and SEQ ID NO 11.

[0097] Fragments that are preferred are those which are binding sites ofthe proteins or protein complexes. For example, the following arepreferred: drug modulatory sites which have been mapped to M1, M2, M2-M3loop, and M3, and the amino terminal sequences.

[0098] Proteins which would result from expression of the nucleic acidmolecules herein disclosed are preferred, with the proteins which wouldresult from expression of the exemplified compounds being mostpreferred. It is understood that proteins which would result fromexpression of allelic variants of the exemplified sequences, as well asproteins which would result from the expression of nucleic acidmolecules which hybridize under stringent hybridization conditions tothe nucleic acid molecules exemplified are within the scope of thepresent invention as well.

[0099] More preferred proteins of the present invention include proteinscomprising amino acid sequences that are at least about 70%, even morepreferably at least about 75%, even more preferably at least about 80%,even more preferably at least about 85%, even more preferably at leastabout 90%, and even more preferably at least about 95%, identical toamino acid sequence exemplified herein. Percent identity can bedetermined use of well-known methods.

[0100] There are also provided recombinant cells comprising the proteinsherein described.

[0101] Also provided are protein homologs of the present invention.Protein homologs can be the result of natural allelic variation ornatural mutation. Protein homologs of the present invention can also beproduced using techniques known in the art including, but not limitedto, direct modifications to the protein or modifications to the geneencoding the protein using, for example, classic or recombinant nucleicacid techniques to effect random or targeted mutagenesis.

[0102] One embodiment of the present invention is a fusion protein thatincludes a protein domain attached to one or more fusion segments.Suitable fusion segments for use with the present invention include, butare not limited to, segments that can: enhance a protein's stabilityand/or assist purification (e.g., by affinity chromatography). Asuitable fusion segment can be a domain of any size that has the desiredfunction (e.g., imparts increased stability, imparts increasedimmunogenicity to a protein, and/or simplifies purification of aprotein). Fusion proteins are preferably produced by culturing arecombinant cell transformed with a fusion nucleic acid molecule thatencodes a protein including the fusion segment attached to either thecarboxyl and/or amino terminal end. Preferred fusion segments include ametal binding domain (e.g., a poly-histidine segment); an immunoglobulinbinding domain (e.g., Protein A; Protein G; T cell; B cell; complementprotein antibody-binding domains); a sugar binding domain (e.g., amaltose binding domain); a “tag” domain (e.g., at least a portion ofβ-galactosidase, a strep tag peptide, other domains that can be purifiedusing compounds that bind to the domain, such as monoclonal antibodies);and/or a linker and enzyme domain (e.g., alkaline phosphatase domainconnected to a protein by a linker). More preferred fusion segmentsinclude metal binding domains, such as a poly-histidine segment; amaltose binding domain; a strep tag peptide; and a phage T7 S10 peptide.

[0103] In one embodiment, an isolated protein of the present inventionis produced by culturing a cell capable of expressing the protein underconditions effective to produce the protein, and recovering the protein.A preferred cell to culture is a recombinant cell of the presentinvention. Effective culture conditions include, but are not limited to,effective media, bioreactor, temperature, pH and oxygen conditions thatpermit protein production. An effective medium refers to any medium inwhich a cell is cultured to produce a protein of the present invention.Such a medium typically comprises an aqueous medium having assimilablecarbon, nitrogen and phosphate sources, and appropriate salts, minerals,metals and other nutrients, such as vitamins. Cells of the presentinvention can be cultured in conventional fermentation bioreactors,shake flasks, test tubes, microtiter dishes, and petri plates. Culturingcan be carried out at a temperature, pH and oxygen content appropriatefor a recombinant cell. Such culturing conditions are within theexpertise of one of ordinary skill in the art.

[0104] Depending on the vector and host system used for production,resultant proteins of the present invention may either remain within therecombinant cell; be secreted into the fermentation medium; be secretedinto a space between two cellular membranes, such as the periplasmicspace in E. coli; or be retained on the outer surface of a cell or viralmembrane. The phrase “recovering the protein”, as well as similarphrases, refers to collecting the whole fermentation medium containingthe protein and need not imply additional steps of separation orpurification. Proteins of the present invention can be purified using avariety of standard protein purification techniques, such as, but notlimited to, affinity chromatography, ion exchange chromatography,filtration, electrophoresis, hydrophobic interaction chromatography, gelfiltration chromatography, reverse phase chromatography, concanavalin Achromatography, chromatofocusing and differential solubilization.Proteins of the present invention are preferably retrieved in“substantially pure” form. As used herein, “substantially pure” refersto a purity that allows for the effective use of the protein as atherapeutic composition or diagnostic. A therapeutic composition foranimals, for example, should exhibit few impurities.

[0105] In addition, recombinant protein can be separated from othercellular proteins by use of an immunoaffinity column made withmonoclonal or polyclonal antibodies specific for herein protein, orpolypeptide fragments of herein protein.

[0106] Also provided are nucleic acid compound which encodes a nematodeneuromuscular junction GABA receptor complex subunits, comprising:

[0107] (a) a nucleic acid sequence which has at least 80% identity toSEQ ID NO 2; wherein said identity can be determined using the DNAsiscomputer program and default parameters;

[0108] (b) a nucleic acid which encodes an amino acid sequence which hasat least 80% identity to SEQ ID NO 1; wherein said identity can bedetermined using the DNAsis computer program and default parameters;

[0109] (c) a nucleic acid sequence which is an allelic variant of SEQ IDNO 2; and

[0110] (d) a nucleic acid sequence fully complementary to a nucleic acidsequence selected from the group consisting of: a nucleic acid sequenceof (a); a nucleic acid sequence of (b); and a nucleic acid sequence of(c).

[0111] Also provided are nucleic acid compounds which encode a nematodeneuromuscular junction GABA receptor complex subunit, comprising anucleic acid sequence selected from the group consisting of:

[0112] (a) a nucleic acid sequence which has at least 80% identity toSEQ ID NO 4; wherein said identity can be determined using the DNAsiscomputer program and default parameters;

[0113] (b) a nucleic acid which encodes an amino acid sequence which hasat least 80% identity to SEQ ID NO 3; wherein said identity can bedetermined using the DNAsis computer program and default parameters;

[0114] (c) a nucleic acid sequence which is an allelic variant of SEQ IDNO 4; and

[0115] (d) a nucleic acid sequence fully complementary to a nucleic acidsequence selected from the group consisting of: a nucleic acid sequenceof (a); a nucleic acid sequence of (b); and a nucleic acid sequence of(c).

[0116] Also provided are nucleic acid compounds which encode a nematodeneuromuscular junction GABA receptor complex, comprising a first andsecond nucleic acid sequence, wherein said first nucleic acid sequenceis selected from the group consisting of:

[0117] (a) a nucleic acid sequence which has at least 80% identity toSEQ ID NO 2; wherein said identity can be determined using the DNAsiscomputer program and default parameters;

[0118] (b) a nucleic acid which encodes an amino acid sequence which hasat least 80% identity to SEQ ID NO 1; wherein said identity can bedetermined using the DNAsis computer program and default parameters;

[0119] (c) a nucleic acid sequence which is an allelic variant of SEQ IDNO 2; and

[0120] (d) a nucleic acid sequence which has at least 80% identity toSEQ ID NO 4; wherein said identity can be determined using the DNAsiscomputer program and default parameters;

[0121] (e) a nucleic acid which encodes an amino acid sequence which hasat least 80% identity to SEQ ID NO 3; wherein said identity can bedetermined using the DNAsis computer program and default parameters;

[0122] (f) a nucleic acid sequence which is an allelic variant of SEQ IDNO 4; and

[0123] wherein said second nucleic acid sequence is selected from thegroup consisting of:

[0124] (a) a nucleic acid sequence which has at least 80% identity toSEQ ID NO 4; wherein said identity can be determined using the DNAsiscomputer program and default parameters;

[0125] (b) a nucleic acid which encodes an amino acid sequence which hasat least 80% identity to SEQ ID NO 3; wherein said identity can bedetermined using the DNAsis computer program and default parameters;

[0126] (c) a nucleic acid sequence which is an allelic variant of SEQ IDNO 4.

[0127] Preferred isolated nucleic acid compound of the present inventionare those which comprise a nucleic acid sequence selected from the groupconsisting of: SEQ ID NO 2; SEQ ID NO 4; and SEQ ID NO 6; SEQ ID NO 8;SEQ ID NO 10; SEQ ID NO 12.

[0128] It is known in the art that there are commercially availablecomputer programs for determining the degree of similarity between twonucleic acid sequences. These computer programs include various knownmethods to determine the percentage identity and the number and lengthof gaps between hybrid nucleic acid molecules. Preferred methods todetermine the percent identity among amino acid sequences and also amongnucleic acid sequences include analysis using one or more of thecommercially available computer programs designed to compare and analyzenucleic acid or amino acid sequences. These computer programs include,but are not limited to, GCG™ (available from Genetics Computer Group,Madison, Wis.), DNAsiS™ (available from Hitachi Software, San Bruno,Calif.) and MacVector™ (available from the Eastman Kodak Company, NewHaven, Conn.). A preferred method to determine percent identity amongamino acid sequences and also among nucleic acid sequences includesusing the Compare function by maximum matching within the program DNAsisVersion 2.1 using default parameters. A nucleic acid sequence of thepresent invention may have at least 75%, preferably 80%, and mostpreferably 95% sequence identity with a nucleic acid molecule in thesequence listing.

[0129] Additional preferred nucleic acid molecules of the presentinvention include an isolated nucleic acid molecule which is at leastabout 50 nucleotides, or at least about 150 nucleotides, comprising anucleic acid sequence that is preferably at least about 45% identical,more preferably about 50% identical, more preferably about 55%identical, more preferably about 60% identical, more preferably about65% identical, more preferably about 70% identical, more preferablyabout 75% identical, more preferably about 80% identical, morepreferably about 85% identical, more preferably about 90% identical andeven more preferably about 95% identical to a nucleic acid sequenceselected from the exemplified sequences. Also preferred are fragments ofany of such nucleic acid molecules. Percent identity may be determinedusing the Compare function by maximum matching within the program DNAsisVersion 2.1 using default parameters.

[0130] The present invention also comprises expression vectors andrecombinant cells comprising the present nucleic acid molecules. Alsoprovided are fusion proteins constructed using the present nucleic acidcompounds.

[0131] Included within the scope of the present invention, withparticular regard to the nucleic acids above, are allelic variants,degenerate sequences and homologues. Allelic variants are well known tothose skilled in the art and would be expected to be found within agiven diploid organism and/or among a group of organisms. The presentinvention also includes variants due to laboratory manipulation, suchas, but not limited to, variants produced during polymerase chainreaction amplification or site-directed mutagenesis. It is also wellknown that there is a substantial amount of redundancy in the variouscodons which code for specific amino acids. Therefore, this invention isalso directed to those nucleic acid sequences which contain alternativecodons which code for the eventual translation of the identical aminoacid. Also included within the scope of this invention are mutationseither in the nucleic acid sequence or the translated protein which donot substantially alter the ultimate physical properties of theexpressed protein. For example, substitution of valine for leucine,arginine for lysine, or asparagine for glutamine may not cause a changein functionality of the polypeptide.

[0132] Knowing the nucleic acid sequences of certain nucleic acidmolecules of the present invention allows one skilled in the art to, forexample, (a) make copies of those nucleic acid molecules, (b) obtainnucleic acid molecules including at least a portion of such nucleic acidmolecules (e.g., nucleic acid molecules including full-length genes,full-length coding regions, regulatory control sequences, truncatedcoding regions), and (c) obtain nucleic acid molecules from otherspecies. Such nucleic acid molecules can be obtained in a variety ofways including screening appropriate expression libraries withantibodies of the present invention; traditional cloning techniquesusing oligonucleotide probes of the present invention to screenappropriate libraries of DNA; and PCR amplification of appropriatelibraries or DNA using oligonucleotide primers of the present invention.Preferred libraries to screen or from which to amplify nucleic acidmolecules include protozoal or mycoplasma libraries as well as genomicDNA libraries. Similarly, preferred DNA sources to screen or from whichto amplify nucleic acid molecules include adult cDNA and genomic DNA.Techniques to clone and amplify genes are disclosed, for example, inSambrook et al., ibid.

[0133] The translation of the RNA into a peptide or a protein willresult in the production of at least a portion of the protein which canbe identified, for example, by the activity of protein or byimmunological reactivity with an anti-protein antibody. In this method,pools of mRNA isolated from protein-producing cells can be analyzed forthe presence of an RNA which encodes at least a portion of the protein.Further fractionation of the RNA pool can be done to purify the hereinprotein RNA from non-protein RNA. The peptide or protein produced bythis method may be analyzed to provide amino acid sequences which inturn are used to provide primers for production of cDNA, or the RNA usedfor translation can be analyzed to provide nucleotide sequences encodingherein proteins and produce probes for the production of cDNA. Thesemethods are known in the art and can be found in, for example, Sambrook,J., Fritsch, E. F., Maniatis, T. in Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. 1989.

[0134] One embodiment of the present invention includes a recombinantvector, which includes at least one isolated nucleic acid molecule ofthe present invention, inserted into any vector capable of deliveringthe nucleic acid molecule into a host cell. Such a vector containsheterologous nucleic acid sequences, that is, nucleic acid sequencesthat are not naturally found adjacent to nucleic acid molecules of thepresent invention and that preferably are derived from a species otherthan the species from which the nucleic acid molecule(s) are derived.The vector can be either RNA or DNA, either prokaryotic or eukaryotic,and typically is a virus or a plasmid. Recombinant vectors can be usedin the cloning, sequencing, and/or otherwise manipulation of the nucleicacid molecules of the present invention.

[0135] One type of recombinant vector, referred to herein as arecombinant molecule, comprises a nucleic acid molecule of the presentinvention operatively linked to an expression vector. The phrase“operatively linked” refers to insertion of a nucleic acid molecule intoan expression vector in a manner such that the molecule is able to beexpressed when transformed into a host cell. As used herein, anexpression vector is a DNA or RNA vector that is capable of transforminga host cell and of effecting expression of a specified nucleic acidmolecule. Preferably, the expression vector is also capable ofreplicating within the host cell. Expression vectors can be eitherprokaryotic or eukaryotic, and are typically viruses or plasmids.Expression vectors of the present invention include any vectors thatfunction (i.e., direct gene expression) in recombinant cells of thepresent invention, including in bacterial, fungal, endoparasite, insect,other animal, and plant cells. Preferred expression vectors of thepresent invention can direct gene expression in bacterial, yeast, insectand mammalian cells and more preferably in the cell types disclosedherein.

[0136] In particular, expression vectors of the present inventioncontain regulatory sequences such as transcription control sequences,translation control sequences, origins of replication, and otherregulatory sequences that are compatible with the recombinant cell andthat control the expression of nucleic acid molecules of the presentinvention. In particular, recombinant molecules of the present inventioninclude transcription control sequences. Transcription control sequencesare sequences which control the initiation, elongation, and terminationof transcription. Particularly important transcription control sequencesare those which control transcription initiation, such as promoter,enhancer, operator and repressor sequences. Suitable transcriptioncontrol sequences include any transcription control sequence that canfunction in at least one of the recombinant cells of the presentinvention. A variety of such transcription control sequences are knownto those skilled in the art. Preferred transcription control sequencesinclude those which function in bacterial, yeast, insect and mammaliancells, such as, but not limited to, tac, lac, trp, trc, oxy-pro,omp/lpp, rrnB, bacteriophage lambda (such as lambda p_(L) and lambdap_(R) and fusions that include such promoters), bacteriophage T7, T7lac,bacteriophage T3, bacteriophage SP6, bacteriophage SP01,metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirussubgenomic promoters (such as Sindbis virus subgenomic promoters),antibiotic resistance gene, baculovirus, Heliothis zea insect virus,vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus,adenovirus, cytomegalovirus (such as intermediate early promoters),simian virus 40, retrovirus, actin, retroviral long terminal repeat,Rous sarcoma virus, heat shock, phosphate and nitrate transcriptioncontrol sequences as well as other sequences capable of controlling geneexpression in prokaryotic or eukaryotic cells. Additional suitabletranscription control sequences include tissue-specific promoters andenhancers as well as lymphokine-inducible promoters (e.g., promotersinducible by interferons or interleukins). Transcription controlsequences of the present invention can also include naturally-occurringtranscription control sequences naturally associated with humans. Thepresent invention also comprises expression vectors comprising a nucleicacid molecule described herein.

[0137] Recombinant DNA technologies can be used to improve expression oftransformed nucleic acid molecules by manipulating, for example, thenumber of copies of the nucleic acid molecules within a host cell, theefficiency with which those nucleic acid molecules are transcribed, theefficiency with which the resultant transcripts are translated, and theefficiency of post-translational modifications. Recombinant techniquesuseful for increasing the expression of nucleic acid molecules of thepresent invention include, but are not limited to, operatively linkingnucleic acid molecules to high-copy number plasmids, integration of thenucleic acid molecules into one or more host cell chromosomes, additionof vector stability sequences to plasmids, substitutions ormodifications of transcription control signals (e.g., promoters,operators, enhancers), substitutions or modifications of translationalcontrol signals (e.g., ribosome binding sites, Shine-Dalgarnosequences), modification of nucleic acid molecules of the presentinvention to correspond to the codon usage of the host cell, deletion ofsequences that destabilize transcripts, and use of control signals thattemporally separate recombinant cell growth from recombinant enzymeproduction during fermentation. The activity of an expressed recombinantprotein of the present invention may be improved by fragmenting,modifying, or derivatizing nucleic acid molecules encoding such aprotein.

[0138] Also provided by the present invention are recombinant cellstransformed with a nucleic acid described herein.

[0139] Transformation of a nucleic acid molecule into a cell can beaccomplished by any method by which a nucleic acid molecule can beinserted into the cell. Transformation techniques include, but are notlimited to, transfection, electroporation, microinjection, lipofection,adsorption, and protoplast fusion. A recombinant cell may remainunicellular or may grow into a tissue, organ or a multicellularorganism. Transformed nucleic acid molecules of the present inventioncan remain extrachromosomal or can integrate into one or more siteswithin a chromosome of the transformed (i.e., recombinant) cell in sucha manner that their ability to be expressed is retained.

[0140] Suitable host cells to transform include any cell that can betransformed with a nucleic acid molecule of the present invention. Hostcells can be either untransformed cells or cells that are alreadytransformed with at least one nucleic acid molecule (e.g., nucleic acidmolecules encoding one or more proteins of the present invention and/orother proteins useful in the production of multivalent vaccines). Hostcells of the present invention either can be endogenously (i.e.,naturally) capable of producing proteins of the present invention or canbe capable of producing such proteins after being transformed with atleast one nucleic acid molecule of the present invention. Host cells ofthe present invention can be any cell capable of producing at least oneprotein of the present invention, and include bacterial, fungal(including yeast), other insect, other animal and plant cells. Preferredhost cells include bacterial, mycobacterial, yeast, parasite, insect andmammalian cells.

[0141] A recombinant cell is preferably produced by transforming a hostcell with one or more recombinant molecules, each comprising one or morenucleic acid molecules of the present invention operatively linked to anexpression vector containing one or more transcription controlsequences. The phrase “operatively linked” refers to insertion of anucleic acid molecule into an expression vector in a manner such thatthe molecule is able to be expressed when transformed into a host cell.

[0142] A recombinant cell of the present invention includes any celltransformed with at least one of any nucleic acid molecule of thepresent invention. Suitable and preferred nucleic acid molecules as wellas suitable and preferred recombinant molecules with which to transformcells are disclosed herein.

[0143] Moreover, there are provided isolated antibodies selective for aGABA receptor complexes of the present invention.

[0144] The present invention also includes isolated (i.e., removed fromtheir natural milieu) antibodies that selectively bind to a portion ofthe protein of the present invention or a mimetope thereof. Binding canbe measured using a variety of methods standard in the art includingenzyme immunoassays (e.g., ELISA), immunoblot assays, etc.; see, forexample, Sambrook et al., ibid. An anti-herein protein antibodypreferably selectively binds to herein protein in such a way as toreduce the activity of that protein. These antibodies may be admixed orconjugated with additional materials, such as cytotic agents or otherantibody fragments.

[0145] Isolated antibodies of the present invention can includeantibodies in a bodily fluid (such as, but not limited to, serum), orantibodies that have been purified to varying degrees. Antibodies of thepresent invention can be polyclonal or monoclonal. Functionalequivalents of such antibodies, such as antibody fragments andgenetically-engineered antibodies (including single chain antibodies orchimeric antibodies that can bind to more than one epitope) are alsoincluded in the present invention.

[0146] A preferred method to produce antibodies of the present inventionincludes (a) administering to an animal an effective amount of aprotein, peptide or mimetope thereof of the present invention to producethe antibodies and (b) recovering the antibodies. In another method,antibodies of the present invention are produced recombinantly usingtechniques as heretofore disclosed to produce proteins of the presentinvention.

[0147] In another embodiment, therapeutic compositions such as theherein described antibodies, nucleic acid compounds and/or the aminoacid compounds (preferrably for immuno-therapy/vaccines) can beformulated in an excipient that the animal to be treated can tolerate.Examples of such excipients include water, saline, Ringer's solution,dextrose solution, Hank's solution, and other aqueous physiologicallybalanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesameoil, ethyl oleate, or triglycerides may also be used. Other usefulformulations include suspensions containing viscosity enhancing agents,such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipientscan also contain minor amounts of additives, such as substances thatenhance isotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer and Tris buffer, while examples ofpreservatives include thimerosal, cresols, formalin and benzyl alcohol.Standard formulations can either be liquid injectables or solids whichcan be taken up in a suitable liquid as a suspension or solution forinjection. Thus, in a non-liquid formulation, the excipient can comprisedextrose, human serum albumin, preservatives, etc., to which sterilewater or saline can be added prior to administration.

[0148] Administration of the compounds can be by a variety of routesknown to those skilled in the art including, but not limited to,subcutaneous, intradermal, intravenous, intranasal, oral, transdermal,intramuscular routes and other parenteral routes.

[0149] In one embodiment of the present methods, a therapeuticcomposition can include an adjuvant. Adjuvants are agents that arecapable of increasing the immune response of an animal to a specificantigen. Protein adjuvants of the present invention can be delivered inthe form of the protein themselves or of nucleic acid molecules encodingsuch proteins using the methods described herein.

[0150] In another embodiment of the present methods, a therapeuticcomposition can include a carrier. Carriers include compounds thatincrease the half-life of a therapeutic composition in the treatedanimal. Suitable carriers include, but are not limited to, polymericcontrolled release vehicles, biodegradable implants, liposomes,bacteria, viruses, other cells, oils, esters, and glycols.

[0151] Another embodiment of the present methods is a controlled releaseformulation that is capable of slowly releasing a composition of thepresent invention into an animal. As used herein, a controlled releaseformulation comprises a composition of the present invention in acontrolled release vehicle. Suitable controlled release vehiclesinclude, but are not limited to, biocompatible polymers, other polymericmatrices, capsules, microcapsules, microparticles, bolus preparations,osmotic pumps, diffusion devices, liposomes, lipospheres, andtransdermal delivery systems. Other controlled release formulations ofthe present invention include liquids that, upon administration to ananimal, form a solid or a gel in situ. Preferred controlled releaseformulations are biodegradable (i.e., bioerodible).

[0152] A preferred controlled release formulation of the presentinvention is capable of releasing a composition of the present inventioninto the blood of an animal at a constant rate sufficient to attaintherapeutic dose levels in the animal. The therapeutic composition ispreferably released over a period of time ranging from about 1 day toabout 12 months, and include release over a 2, 3, 4, 5, 6, 7 day througha 30 day time period.

[0153] Acceptable protocols to administer therapeutic compositions ofthe present invention in an effective manner include individual dosesize, number of doses, frequency of dose administration, and mode ofadministration. Determination of such protocols can be accomplished bythose skilled in the art. A suitable single dose is a dose that iscapable of protecting (i.e., preventing or treating) an animal fromdisease when administered one or more times over a suitable time period.The need for additional administrations of a therapeutic composition canbe determined by one of skill in the art in accordance with the givencondition of a patient.

[0154] Pharmaceutically useful compositions comprising herein proteins,may be formulated according to known methods such as by the admixture ofa pharmaceutically acceptable carrier, or by modification withadditional chemical moieties so as to form a chemical derivative.Examples of such carriers, modifications and methods of formulation maybe found in Remington's Pharmaceutical Sciences. To form apharmaceutically acceptable composition suitable for effectiveadministration, such compositions will contain an effective amount ofthe protein or DNA.

[0155] The present invention also has the objective of providingsuitable topical, oral, systemic and parenteral formulations of thepharmaceutical compounds herein provided. The formulations can beadministered in a wide variety of therapeutic dosage forms inconventional vehicles for administration. For example, the compounds canbe formulated for oral administration in the form of tablets, capsules(each including timed release and sustained release formulations),pills, powders, granules, elixirs, tinctures, solutions, suspensions,syrups and emulsions, or by injection. Likewise, they may also beadministered intravenously (both bolus and infusion), duringangioplasty/catheterization, intraperitoneally, subcutaneously,topically with or without occlusion, or intramuscularly, all using formswell known to those of ordinary skill in the pharmaceutical arts.

[0156] A molecule can be combined with a buffer in which the molecule issolubilized, and/or with a carrier. Suitable buffers and carriers areknown to those skilled in the art. Examples of suitable buffers includeany buffer in which a molecule can function to inhibit its targetenzyme(s), such as, but not limited to, phosphate buffered saline,water, saline, phosphate buffer, bicarbonate buffer, HEPES buffer(N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid buffered saline),TES buffer (Tris-EDTA buffered saline), Tris buffer and TAE buffer(Tris-acetate-EDTA). Examples of carriers include, but are not limitedto, polymeric matrices, toxoids, and serum albumins, such as bovineserum albumin.

[0157] For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water, and the like. Moreover, when desired or necessary,suitable binders, lubricants, disintegrating agents and coloring agentscan also be incorporated into the mixture. Suitable binders include,without limitation, starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes and the like. Lubricants used in these dosageforms include, without limitation, sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride,and the like. Disintegrators include, without limitation, starch, methylcellulose, agar, bentonite, xanthan gum and the like.

[0158] For liquid forms the active drug component can be combined insuitably flavored suspending or dispersing agents such as the syntheticand natural gums, for example, tragacanth, acacia, methylcellulose andthe like. Other dispersing agents which may be employed include glycerinand the like. For parenteral administration, sterile suspensions andsolutions are desired. Isotonic preparations which generally containsuitable preservatives are employed when intravenous administration isdesired.

[0159] Topical preparations containing the active drug component can beadmixed with a variety of carrier materials well known in the art, suchas, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and Eoils, mineral oil, PPG2 myristyl propionate, and the like, to form,e.g., alcoholic solutions, topical cleansers, cleansing creams, skingels, skin lotions formulations. The compounds of the present inventioncan also be administered in the form of liposome delivery systems, suchas small unilamellar vesicles, large unilamellar vesicles andmultilamellar vesicles. Liposomes can be formed from a variety ofphospholipids, such as cholesterol, stearylamine orphosphatidylcholines.

[0160] The compounds of the present invention may also be coupled withsoluble polymers as targetable drug carriers. Such polymers can includepolyvinyl-pyrrolidone, pyran copolymer,polyhydroxypropylmethacryl-amidephenol,polyhydroxy-ethylaspartamidephenol, or polyethyleneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the compounds of thepresent invention may be coupled to a class of biodegradable polymersuseful in achieving controlled release of a drug, for example,polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates andcross-linked or amphipathic block copolymers of hydrogels.

[0161] In addition, there are provided methods to determine a testsubstance's ability to interact with a nematode neuromuscular junctionGABA receptor complex, comprising contacting a receptor complex of thepresent invention with a test substance and determining whether saidtest substance and said receptor complex interact.

[0162] Other methods include those to detect GABA receptors in a testsample, comprising: (a) immobilizing a test sample on a substrate; (b)contacting the test sample with an antibody of the present inventionunder conditions suitable for formation of a GABA receptor:antibodycomplex bound to the substrate; (c) removing non-bound material from thesubstrate under conditions that retain GABA:antibody complex binding tothe substrate; and (d) detecting the presence of the GABAreceptor:antibody complex.

[0163] As used herein, the term “contacting” refers to combining ormixing ingredients, as all of those terms are known in the art.Conditions suitable to form a complex; such conditions (e.g.,appropriate concentrations, buffers, temperatures, reaction times) aswell as methods to optimize such conditions are known to those skilledin the art, and examples are disclosed herein. Complex formationconditions are also disclosed in the Examples.

[0164] In one embodiment, a test substance of the present methodincludes a biological sample from an animal. A suitable biologicalsample includes, but is not limited to, a bodily fluid composition or acellular composition. A bodily fluid refers to any fluid that can becollected (i.e., obtained) from an animal, examples of which include,but are not limited to, blood, serum, plasma, urine, tears, aqueoushumor, cerebrospinal fluid (CSF), saliva, lymph, nasal secretions, milkand feces.

[0165] Detection can be accomplished in a variety of ways including, butnot limited to use of one or more of the following assays: anenzyme-linked immunoassay, a radioimmunoassay, a fluorescenceimmunoassay, a chemiluminescent assay, a lateral flow assay, anagglutination assay, a particulate-based assay (e.g., using particulatessuch as, but not limited to, magnetic particles or plastic polymers,such as latex or polystyrene beads), an immunoprecipitation assay, aBioCore™ assay (e.g., using colloidal gold) and an immunoblotting assay(e.g., a western blot). Such assays are well known to those skilled inthe art. Assays can be used to give qualitative or quantitative resultsdepending on how they are used. Some assays, such as agglutination,particulate separation, and immunoprecipitation, can be observedvisually (e.g., either by eye or by a machines, such as a densitometeror spectrophotometer) without the need for a detectable marker. In otherassays, conjugation (i.e., attachment) of a detectable marker or to areagent that selectively binds to the protein or nucleic acid or to themolecule being detected (described in more detail below) aids indetection. Examples of detectable markers include, but are not limitedto, a radioactive label, an enzyme, a fluorescent label, achemiluminescent label, a chromophoric label or a ligand. A ligandrefers to a molecule that binds selectively to another molecule.Preferred detectable markers include, but are not limited to,fluorescein, a radioisotope, a phosphatase (e.g., alkaline phosphatase),biotin, avidin, a peroxidase (e.g., horseradish peroxidase) andbiotin-related compounds or avidin-related compounds (e.g., streptavidinor ImmunoPure® NeutrAvidin available from Pierce, Rockford, Ill.).According to the present invention, a detectable marker can be connectedto a molecule using, for example, chemical conjugation or recombinantDNA technology (e.g., connection of a fusion segment such as thatdescribed herein for a metal binding domain; an immunoglobulin binding;a sugar binding domain; and a “tag” domain).

[0166] In one embodiment, the method can be accomplished in solution. Inanother embodiment, one or more ingredients are immobilized on (e.g.,coated onto) a substrate. Immobilization techniques are known to thoseskilled in the art. Suitable substrate materials include, but are notlimited to, plastic, glass, gel, celluloid, paper, PVDF(poly-vinylidene-fluoride), nylon, nitrocellulose, and particulatematerials such as latex, polystyrene, nylon, nitrocellulose, agarose andmagnetic resin. Suitable shapes for substrate material include, but arenot limited to, a well (e.g., microtiter dish well), a plate, adipstick, a bead, a lateral flow apparatus, a membrane, a filter, atube, a dish, a celluloid-type matrix, a magnetic particle, and otherparticulates. A particularly preferred substrate comprises an ELISAplate, a dipstick, a radioimmunoassay plate, agarose beads, plasticbeads, latex beads, immunoblot membranes and immunoblot papers. In oneembodiment, a substrate, such as a particulate, can include a detectablemarker.

[0167] A preferred method to detect is via generation or inhibition ofan electrical, preferrably a chloride ion, current in a frog oocyteassay. The examples, as well as many journal articles and textbooks,describe frog oocyte assays of this nature.

[0168] The following examples illustrate the present invention without,however, limiting it. It is to be noted that the Examples include anumber of molecular biology, microbiology, immunology and biochemistrytechniques considered to be known to those skilled in the art.Disclosure of such techniques can be found, for example, in Sambrook etal., ibid., and related references.

EXAMPLE 1 Experimental Procedures

[0169]C. elegans Strains

[0170] unc-49 strains and corresponding alleles used in this study areas follows: CB382: unc-49(e382), CB407: unc-49(e407), EG1232:unc-49(e468), CB641: unc-49(e641), CB929: unc-49(e929), MT2976:unc-49(n1324), MT3123: unc-49(n1324n1345), MT6224: unc-49(n2392).MT6225: unc-49(n2393) is likely to be a re-isolate of n2392 since themutations are identical. The n1324 allele was isolated from MT2879, inwhich Tc1 transposons are active. The above list represents all unc-49alleles isolated to date.

[0171]C. elegans Transformation

[0172] Transformation was performed by microinjection of plasmid andcosmid DNA into the C. elegans germline according to Mello, C. C.,Kramer, J. M., Stinchcomb, D., and Ambros, V. (1991). Efficient genetransfer in C. elegans: extrachromosomal maintenance and integration oftransforming sequences. EMBO J. 10, 3959-3970. T21C12 and T21C12ΔMluwere injected at 80 ng/μl into unc-49(e382); lin-15(n765ts). pEK1, aplasmid that contains the wild-type lin-15 gene (S. G. Clark and X. Lu,personal communication; Clark, S. G., Lu, X., and Horvitz, H. R. (1994).The Caenorhabditis elegans locus lin-15, a negative regulator of atyrosine kinase signaling pathway, encodes two different proteins.Genetics 137, 987-997), was co-injected at 80 ng/μl as aco-transformation marker. Progeny of injected animals were raised at therestrictive temperature for lin-15(n765ts), and successfully transformedanimals were recognized by their nonMuv phenotype.

[0173] T21C12 and T21C12ΔMlu only contain 290 base pairs upstream of thestart codon. These plasmids were able to rescue the strong shrinkerphenotype of unc-49, but they were incapable of complete rescue.Transformed animals could not move in a straight line. Instead, theycurved dorsally, suggesting overexpression of GABA receptors on theventral side relative to the dorsal side. Complete rescue was obtainedby co-injecting two overlapping linear DNA fragments which recombined inthe germline to form the complete unc-49 locus with an additional 4 kbof 5′ flanking DNA. One fragment was a genomic PCR fragment containingthe 4 kb 5′ flanking DNA and 4 kb of the 5′ end of the T21C12 insert(amplified with primers 40 and 110). The other fragment was agel-purified Spe I-Mlu I fragment of T21C12. The overlap between thesetwo fragments was 970 bases. Fragments were injected at roughly 10 ng/μleach, along with 40 ng/μl pEK1 and 40 ng/μl 1 kb ladder (Gibco/BRL).Transformed animals from these injections were fully-rescued. Thismethod was employed because constructs containing the 4 kb of 5′flanking DNA were unstable and could not be maintained in bacterialhosts. This method was also used to demonstrate that fragments lackingthe UNC-49A or UNC-49C open reading frames were also capable of unc-49rescue. The UNC-49A open reading frame was eliminated by Klenow-fillingthe Nde I site near the UNC-49A M3 domain, and the UNC-49C open readingframe was eliminated by deleting a fragment between two Nru I sites,which included all of the UNC-49C M1 domain.

[0174] cDNA Analysis

[0175] This section is an overview of the experiments that led to theisolation of UNC-49A, UNC-49B, UNC-49C and UNC-49Cshort cDNA clones.Details of individual experimental procedures are presented insubsequent sections.

[0176] UNC-49A: The first UNC-49A cDNA clones were isolated from thecDNA library supplied by P. Okkema, probed with a mixture of labeled PCRfragments generated using primers 7 (corresponding to the conserveddisulfide-bonded loop) and 8 (corresponding to repeat A, M4), andprimers 5 (repeat B, M1) and 6 (repeat B, M4). Four partially-splicedUNC-49A cDNA clones were isolated. We then performed an RT-PCRexperiment to isolate additional UNC-49A clones. We used first-strandcDNA prepared using polyA-selected C. elegans RNA (see ‘Preparation offirst strand cDNA’ section). PCR was performed in two rounds. In thefirst round, primer 68 (conserved amino-terminal domain) was paired withprimer 93 (repeat A, M4). This reaction produced a product of about 1 kbthat was cloned using the TA cloning kit (Invitrogen). One μl of thisreaction was re-amplified using the nested primers 73 and 94. Thisreaction produced an abundant 1 kb product that was likewise TA-cloned.Colonies from both ligations were analyzed by colony hybridization usingthe partial UNC-49A cDNA isolated above, and 96 positive colonies werepicked and analyzed by double-digestion with Rsa I and Not I restrictionenzymes (GIBCO/BRL). Based on unique restriction patterns, 14 cloneswere sequenced and five of these corresponded to fully-spliced UNC-49AmRNA. Six of the remaining clones contained unspliced introns oraberrant splice patterns that interrupted the UNC-49A open readingframe, two clones contained internal deletions, and one clone containednon-unc-49 sequences.

[0177] UNC-49B: Three UNC-49B cDNA clones were isolated from the cDNAlibrary provided by R. Barstead. Two of these clones were identical andtherefore probably not independent. Additional UNC-49B cDNA clones wereisolated in two RT-PCR experiments. In the first, first-strand cDNAprepared from total C. elegans RNA amplified as described for UNC-49A,using primers 5 (repeat B, M1) and 6 (repeat B, M4). Seventeen cloneswith inserts were further analyzed by double-digesting with Rsa I andNot I restriction enzymes (GIBCO/BRL). Using these enzymes, we were ableto discriminate between the three UNC-49B isoforms. This analysis showedthat 7 out of 17 corresponded to UNC-49B.1; 9/17 corresponded toUNC-49B.2; and 1/17 corresponded to UNC-49B.3. In the second RT-PCRexperiment, first-strand cDNA prepared using polyA-selected C. elegansRNA was amplified using primers 68 and 74 (repeat B, M4), SL1/74, andSL2/74 (see FIG. 2). Next, 1 μl of each reaction was re-amplified in asecond round of PCR reactions using the nested primer pairs 73/6, SL1/6,and SL2/6. Each of these reactions produced plainly visible bands whenanalyzed by agarose gel electrophoresis, and reaction products werecloned using the TA Cloning kit (Invitrogen). Transformations wereanalyzed by colony hybridization (according to Ausubel, F. M., Brent,R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., andStruhl, K. (1995). Current Protocols in Molecular Biology, K. Janssen,ed.: John Wiley and Sons, Inc.) using Duralon nylon filters(Stratagene). An UNC-49B cDNA fragment was used as a probe after it hadbeen gel-purified away from vector sequences using the QIAquick GelExtraction Kit (Qiagen), and labeled by random priming (specificactivity>1×10⁸ cpm/μg). Positive colonies were identified using aPhosphorimager (Applied Biosystems) and 9-10 positive colonies werepicked from each plate (The SL2/6 PCR reaction was performed twice, anda total of 9 positive colonies were picked from these two trials). Eachclone was then subjected to double-digestion with Rsa I and Not Irestriction enzymes, and clones with unique restriction patterns wereidentified. Clones with unique restriction patterns were sequenced. Weisolated two UNC-49B.1 and one UNC-49B.2 cDNA clones.

[0178] UNC-49C: The isolation of UNC-49C and UNC-49B cDNA clones wasperformed simultaneously. Only details specific to the isolation ofUNC-49C clones are noted here. Two independent UNC-49C cDNA clones wereisolated from the library supplied by R. Barstead. RT-PCR analysis oftotal C. elegans RNA was performed using primers 1 (N-terminus of repeatC) and 4 (repeat C, M4). Fourteen clones contained inserts whichrepresented a single size class. One of these was sequenced, and foundto correspond to the UNC-49C splicing pattern. RT-PCR of polyA-selectedC. elegans RNA was performed as described for UNC-49B using primers 75(repeat C, M4) and 4. We sequenced two UNC-49C cDNA clones isolated inthis experiment.

[0179] UNC-49Cshort: Nine UNC-49Cshort clones were isolated from thecDNA library supplied by R. Barstead. The RT-PCR analysis ofpolyA-selected C. elegans RNA described above should have detectedUNC-49Cshort mRNA had it contained trans-spliced SL1 or SL2 leadersequences. We did not isolate SL-spliced UNC-49Cshort cDNA clones.However, we isolated other cDNA clones in which SL1 or SL2 sequenceswere spliced to internal introns. Because such splices are likely to berare splicing errors, our protocols appear to be very sensitive. Thusthe absence of SL1 or SL2 product indicates that these are not normallyproduced.

[0180] Summary Statistics: Twenty-seven cDNA clones were isolated fromthe library supplied by R. Barstead. Twenty-three of these correspondedto unc-49 sequences, and 14 were of sufficient length that they could begrouped into either the UNC-49B, UNC-49C or UNC-49Cshort class.Ninety-six clones were isolated in the RT-PCR analysis of total C.elegans RNA, and 175 clones were isolated in the RT-PCR analysis ofpolyA-selected C. elegans RNA. Ninety-six of these were generated byUNC-49A-specific primers, and 79 were generated by primers specific forUNC-49B and UNC-49C. Eighteen of these 79 clones were sequenced.

[0181] Polymerase Chain Reaction

[0182] Reactions were carried out using the PTC-100 or PTC-200 thermalcyclers (MJ Research). We used either Taq DNA polymerase (GIBCO/BRL) orthe Expand Long Template PCR system (Boehringer). Reaction conditionsmatched those suggested by the enzyme manufacturers, unless otherwisenoted. Details of individual PCR experiments are as follows. 1)Reactions containing primer pairs 7/8, 5/6 or 1/4 using eitherreverse-transcribed C. elegans total RNA (see ‘Preparation offirst-strand cDNA’ section) or purified T21C12 cosmid DNA as templatewere cycled with the following parameters: 94° C., 30″; 55° C., 30″; 72°C., 1′30″; for 30 cycles, followed by a 7′ incubation at 72° C. Taqpolymerase was used for these reactions. 2) For PCR analysis ofbacterial colonies, bacterial cells were transferred to 50 μl of LBbroth containing 50 μg/ml ampicillin. 25 μl of this culture was diluted1:4 with H₂O and boiled in a PCR reaction tube for 5 minutes using athermal cycler set at 100° C. 10 μl of boiled cell suspension was usedfor subsequent PCR reactions in a 50 μl volume using Taq polymerase andcycling parameters described for (1) above, for 25 cycles. Taqpolymerase was used for these reactions as well. 3) PCR reactions onlambda phage cDNA clones was performed as follows: Phage supernatantswere incubated for 5′ at 100° C. PCR with Taq polymerase was thenperformed as described for (1) above except that an extension time of 7′was used, for 35 cycles. After 20 cycles of this program had beencompleted, an additional 0.5 μl of Taq polymerase was added to eachreaction and the reaction was allowed to proceed for the remaining 15cycles. 4) PCR reactions using reverse-transcribed polyA-selected RNA(see ‘Preparation of first-strand cDNA’ section) or purified genomic DNAfor sequencing (see ‘Genomic Sequencing’) were performed as follows:Following an initial 94° C., 2′ denaturation, samples were subjected to10 cycles of 94° C., 10″; 55° C., 30″; 68° C., 2′; followed by 20-25cycles of 94° C., 10″; 55° C., 30″; 68° C., 2′20″ plus 20 additionalseconds each cycle. The final step was a 7′ incubation at 68° C. TheExpand Long Template PCR System was used for these reactions, and inexperiments using reverse-transcribed poly-A+RNA as the template, theMg2+concentration was increased to 2.5 mM. The sequence of all PCRprimers is shown in the table below: primer sequence 1 atg tgt tca gatgcg tat tcg 4 gat gaa aac aag agg aaa gcg 5 ctg atc gtc acc ata tct tgg6 aag aca atg gga aac cgt atc 7 tgt cca atg gac ctg aag ctg 8 cgg cgtatt cta gaa gtg aac 19 tgg agc ccg tca gta tcg gcg 20 gta gcg acc ggcgct cag ctg 37 atc ccc agc gcc tcc ccg tta 38 ttt ttg cct gtt ttt gtcgcc 39 ata gtc ata aat gga ccc gcg 40 ctc gga aat aat gtg cat gaa 41 ttcaca cat ggt gca tcg aag 42 gct agt gtg ata agt gct gtg 45 cga ttt tctcag tat gca cgg 46 att ttc gca cca cac ctt ctc 47 tat gtc gca aaa ttcgac gcc 48 gat gaa gtg ctg gca agt gtc 68 cac att aga ctt cta cat gcg 73aaa cgt ggc aag acc ctc gac 74 cca gta gac tat att gaa gat 75 agc cagaag aga gtg ttg aac 83 ata cca tca tga agc aga cac 93 atg aag tag gcccag tag ccg 94 gta gcc gac gtt gaa gag cac 110 atg gtg gtt ttg ttc ccctcc SL1 ggt tta att acc caa gtt tga g SL2 ggt ttt aac cca gtt act caa gM13F cgc cag ggt ttt ccc agt cac gac M13R tca cac agg aaa cag cta tga c

[0183] Library Screening

[0184] Two different cDNA libraries were screened. The first, preparedusing poly-A+C. elegans RNA and the Lambda Zap vector (Stratagene), wasa kind gift of Dr. R. Barstead Barstead, R. J., and Waterston, R. H.(1989). The basal component of the nematode dense-body is vinculin. J.Biol. Chem. 264, 10177-10185). This library (350,000 plaques) wasscreened using Duralon nylon filters (Stratagene) according to themanufacturer's instructions using three PCR products roughlycorresponding to the transmembrane domains of C-terminal repeat A, B andC as probes. These fragments were generated using primer pairs 7/8, 5/6,1/4 respectively. Probes were labeled to greater than 1×10⁸ cpm/μg byrandom priming and combined in equal amounts in the hybridizationmixture. Inserts from positive clones were excised using the ExAssisthelper phage/SOLR strain system (Stratagene).

[0185] The second library, prepared from oligo U-selected C. elegansmRNA and the lambda GT11 vector, was kindly supplied by Dr. P. Okkema(Okkema, P. G., and Fire, A. (1994). The Caenorhabditis elegans NK-2class homeoprotein CEH-22 is involved in combinatorial activation ofgene expression in pharyngeal muscle. Development 120, 2175-2186). Thislibrary (400,000 plaques) was screened as described above, except thatthe C-terminal repeat C probe was omitted. Inserts from positive cloneswere PCR amplified using primers 19 and 20 and cloned using the TACloning kit (Invitrogen). Growing and plating of recombinant phage, andidentification of positive plaques was performed according to standardtechniques as described in Ausubel, F. M., Brent, R., Kingston, R. E.,Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1995).Current Protocols in Molecular Biology, K. Janssen, ed.: John Wiley andSons, Inc.).

[0186] Preparation of First-strand cDNA

[0187] First-strand cDNA was prepared in two different ways. First,total C. elegans RNA (D. P. Morse) was reverse-transcribed usingoligo-dT primers (12-18 nucleotides in length) and Superscript IIreverse transcriptase (GIBCO/BRL), according to the protocol suppliedwith the enzyme. Second, C. elegans RNA (D. P. Morse) was first polyAselected, and then reverse transcribed. 30 μg of RNA was dissolved in 10μl H₂O and 20 μl TE/KCl (10 mM tris, 1 mM EDTA, 1M KCl, pH7) was added.Then, 100 μl Dynabeads oligo(dT)₂₅(Dynal) was placed in a separate tube,and liquid was removed after magnetic beads were immobilized using theMPC-1 magnet (Dynal). Beads were resuspended in 100 μl TE/KCl, liquidwas removed, and the RNA solution was added. These components were thenheated to 70° C. and slowly cooled to room temperature over 10 minutesusing a thermal cycler. Beads were immobilized with the magnet and thesupernatant was removed. Beads were then resuspended in 50 μl reversetranscriptase reaction mix. This mix contained the following: 2.5 μl 200mM Tris-HCl, pH 8.3, 1.0M KCl; 2.5 μl 30 mM MgCl₂, 15 mM MnSO₄; 20 μldNTP solution (2.5 mM dATP, dCTP, dGTP, dTTP; Pharmacia); 1.0 μl 40 U/μlRNasin (Boeringer); 2.0 μl 200 U/μl SuperScript II (GIBCO/BRL); 5.0 μl 1U/μl Retrotherm RT (Epicentre Technologies); 17 μl H₂O. Reaction wascarried out at 40° C. for 30 minutes, followed by 70° C. for one hour.Beads were then immobilized with the magnet, liquid was removed, beadswere washed with 100 μl TE (10 mM tris, 1 mM EDTA pH 7), liquid wasremoved, and beads were resuspended in 100 μl TE. One μl of suspendedbeads was used as template in subsequent PCR experiments.

[0188] Northern Analysis

[0189] N2 worms were grown on NGM plates containing 2% agarose (FMC)instead of agar, seeded with HB101 bacteria. Worms were harvested in M9buffer before the plates starved out. RNA was isolated using the directphenol extraction method, except that one chloroform extraction was usedin place of the three ether extractions. PolyA+RNA was purified fromtotal RNA (75 μg per lane) using Oligo-dT Dynabeads (Dynal) according tomanufacturer's instructions. RNA was eluted from beads directly intoNorthern loading buffer. Samples were run on a 1.2%formaldehyde-containing MOPS/EDTA agarose gel and transferred toZeta-probe nylon membranes (Bio-Rad) by capillary transfer usingstandard techniques. Blots were probed with labeled cDNA fragments (10⁸cpm/μg), which specifically hybridized to the UNC-49A (Rsa I-Eco RIfragment of the 7/8 PCR fragment), UNC-49B (5/6 PCR fragment) andUNC-49C (1/4 PCR fragment) mRNAs. Blots were reprobed with an act-1probe (M. Horner) to normalize unc-49 signals for variations in RNAloading and transfer. Band intensity was quantified using aPhosphorimager (Applied Biosystems).

[0190] Computer Sequence Analysis

[0191] Multiple sequence alignments were performed using the Pileupprogram in the Genetics Computer Group software package, version 9.0.Sequences used in the alignment, and their accession numbers, are listedbelow. Rat GABA_(A) receptor subunits α1 (SwissProt: p18504), α2(SwissProt: p23576), α3 (SwissProt: p20236), α4 (SwissProt: p28471), α5(SwissProt: p19969), α6 (SwissProt: p30191), β1 (SwissProt: p15431), β2(SwissProt: p15432), β3 (SwissProt: p15433), γ1 (SwissProt: p23574), γ2(SwissProt: p18508), γ3 (SwissProt: p28473), δ (SwissProt: p18506); RatGABA_(C) receptor subunits ρ1 (SwissProt: p50572), ρ2 (SwissProt:p47742), ρ3 (SwissProt: p50573); Rat glycine receptor subunits α1(SwissProt: p07727), α2 (SwissProt: p22771), α3 (SwissProt: p24524), β(SwissProt: p20781); Human GABA_(A) receptor ε subunit (gb:U66661),Drosophila melanogaster rdl gene product (SwissProt: p25123), DrosophilaGABA receptor β subunit (SwissProt: q08832); lymnaea stagnalis GABAreceptor β subunit (SwissProt: p26714); and avermectin-sensitiveglutamate-gated chloride channel α subunit (pir2:s50864), β subunit(gb:u14525). Alignments were performed with full-length subunits.Alignments of representative GABA receptor subunits used to establishconservation shown in FIG. 4B were performed using the Clustal alignmentmethod within the MegAlign program of the DNAstar sequence analysispackage (DNASTAR). The rat α1, β1, γ1, δ, and ρ1 GABA receptor subunits,the human ε1 GABA receptor subunit, and drososphila rdl protein wereused for this alignment. Signal peptide cleavage sites were predictedusing the PSORT program (K. Nakai, Osaka University). Consesusphosphorylation sites were identified using the ppsearch program (EMBLdata library).

[0192] Genomic Southern Blot Analysis

[0193] Preparation of genomic DNA and Southern blot analysis wereperfomed according to standard techniques using Zeta-probe nylonmembranes (Bio-Rad). Blots were probed with a mixture of three labeledfragments: (1) an Eco RI fragment which includes bases 1043 to 2983 ofT21C12, (2) a genomic PCR product (see ‘Polymerase Chain Reaction’section in Experimental Procedures) generated using primers 7 and 8(FIG. 2A), (3) a second Eco RI fragment which includes bases 8968 to12054 of T21C12. Each fragment was labeled by random priming to aspecific activity of >10⁸ cpm/μg. Prehybridization, hybridization andwashing (high stringency) were perfomed according to manufacturer'sinstructions. Blots were visualized by autoradiography or by using aPhosphorimager (Applied Biosystems).

[0194] DNA Sequencing

[0195] Sequencing of cDNA clones was carried out using an ABI automatedDNA sequencing apparatus at the Sequencing Core Facility, University ofUtah. Genomic sequencing was performed on genomic PCR fragmentscorresponding to UNC-49B using the ThermoSequenase cycle sequencing kit(Amersham).

[0196] GFP Constructs

[0197] The S65C variant of GFP containing 3 introns (1997 Fire vectorkit) was cloned into the T21C12ΔMlu construct such that GFP wasinserted, in frame, into the large intracellular loop of one subunit,while the other subunits were wild-type. UNC-49A was tagged by insertinga Klenow-filled Eco RV to Xba I fragment of pPD103.87 into a T4DNA-polymerase-treated Bsm I site. UNC-49B was tagged by inserting aKlenow-filled Cla I to Bam HI fragment of pPD102.33 into a Bsa BI site.UNC-49C was tagged by inserting a Klenow-filled Cla I to Not I fragmentof pPD103.87 into a T4 DNA-polymerase-treated Bsm I site. Tospecifically tag the putative UNC-49Cshort subunit, the Spe I sitewithin the common amino terminus was Klenow-filled in the UNC-49C-taggedconstruct. To generate transgenic lines expressing the GFP-taggedsubunits, unc-49(e382);lin-15(n765ts) worms were injected with linearfragments of the GFP-tagged constructs and genomic PCR fragmentscontaining 5′ flanking DNA as described above. A slight variation wasused to generate UNC-49B::GFP and UNC-49Cshort::GFP lines: Instead ofco-injecting a Spe I-Mlu I unc-49 fragment with a 110/40 genomic PCRproduct, we co-injected an Afl II-Mlu I unc-49 fragment with a 110/38genomic PCR fragment. This pair of fragments contained 450 base pairs ofoverlapping DNA. As a control, the Afl II-Mlu I fragment of theunmodified T21C12 cosmid was injected with, and without, the 5′ genomicfragment: Rescue of unc-49 required the 5′ genomic fragment.UNC-49B::GFPΔC was constructed by Klenow-filling a Bgl II site nearUNC-49C M1, and UNC-49C::GFPΔB was constructed by Klenow-filling a BsiWI site near UNC-49B M1. These constructs were injected intounc-49(e382);lin-15(n765ts) mutants as circular cosmids, as describedfor the original injections of T21C12 and T21C12ΔMlu. Fluorescence intransformed animals was strong in the ventral cord but weak or absent inthe dorsal cord, suggesting that elements required for dorsal expressionare contained within the 4 kb of 5′ flanking DNA. We confirmed thisobservation by injecting the intact UNC-49B::GFP and UNC-49C::GFPconstructs as circular cosmids, in the absence of the 4 kb of 5′flanking DNA. Transformants from these injections also showed muchstronger GFP fluorescence in the ventral cord than in the dorsal cord.

[0198] Electrophysiology

[0199] Oocytes were removed from Xenopus laevis frogs anesthetized byimmersion in 0.2% tricaine for 15-30 minutes. Harvested ovarian lobeswere defolliculated by incubation in 2 mg/ml of collagenase (Type IA,Sigma) for 2 hours at room temperature on an orbital shaker incalcium-free ND-96 solution containing in mM: 96 NaCl, 2 KCl, 1 MgCl₂,and 5 HEPES (pH=7.6). The oocytes were rinsed 5-6 times with a Barth'ssolution that contained (in mM): 88 NaCl, 1 KCl, 0.41 CaCl₂, 0.33Ca(NO₃)₂, 1 MgSO₄, 2.4 NaHCO₃ and 10 HEPES (pH=7.4), and selected stageV-VI oocytes were stored at 18 C in Barth's solution supplemented with 1mM Na-Pyruvate, 0.01 mg/ml gentamycin and an antibiotic-antimycoticsolution containing 100 units/ml of penicillin, 100 μg/ml streptomycinand 0.25 μg/ml of Amphotericin B (Gibco BRL).

[0200] Oocytes were injected with cRNA (prepared using the mMessagemMachine kit, Ambion) into the cytoplasm 24 hours later. Glass capillarytubes (World Precision Instruments) were pulled to a fine tip on avertical micropipette puller (David Kopf) and broken back to an outsidediameter of 21 μm. RNA stocks were diluted to a final concentration of0.75 μg/μl and injected into the oocytes (32 nl) with a microinjector(World Precision Instruments).

[0201] Electrophysiological recordings were performed 3-7 days followinginjection and were carried out at room temperature in a control Ringerssolution containing in mM: 115 NaCl, 2.5 KCl, 1.0 BaCL₂, and 10 HEPES(pH=7.4). Two electrode voltage clamp recordings were obtained with aGeneclamp amplifier (Axon Instruments) using 3 mM KCl-filledmicroelectrodes (1-5 M). Recordings were carried out at a holdingpotential of −60 mV. Concentration-effect data were fit to the logisticequation:

I=I _(max)/{1+(EC₅₀/[agonist])^(nH)},

[0202] where I_(max) is the maximal current, [agonist] is theconcentration, EC₅₀ is the concentration of agonist resulting in halfmaximal activation and n_(H) is an empirical parameter describing thesteepness of fit and having the same meaning as the Hill coefficient.NFIT (Island Products) was used for non-linear curve fitting. Data arepresented as the mean S.E.M.; n is the number of oocytes tested. Allcombinations of subunits were tested in parallel in at least twoindependent experiments (at least 4 oocytes for each combination ofmRNAs per experiment). The effect of incorporating the UNC-49C subunitwas consistent from one experiment to another. Single channel recordingswere performed as described in except that 1 mM GABA was appliedcontinuously. Single channel conductance was determined by fittingGaussian curves to all points histograms.

EXAMPLE 2 Results

[0203] Structure of the unc-49 Locus

[0204] We cloned unc-49 using standard microinjection-rescue techniquesdescribed by Mello, C. C., Kramer, J. M., Stinchcomb, D., and Ambros, V.(1991), Efficient gene transfer in C. elegans: extrachromosomalmaintenance and integration of transforming sequences. EMBO J. 10,3959-3970. Genetic map data indicated that unc-49 was located onchromosome III, between lin-19 and mel-23. One cosmid in this region,T21C12, contained a predicted 12 kb open reading frame (T21C12.1) withsignificant similarity to GABA_(A) receptor subunits as described inWilson, R. Et al. (1994). 2.2 Mb of contiguous nucleotide sequence fromchromosome III of C. elegans . Nature 368, 32-38. This cosmid wasinjected into unc-49(e382) animals, and two stable lines wereestablished which rescued the unc-49 shrinker phenotype. A constructcontaining only the T21C12.1 open reading frame (T21C12ΔMlu) alsorescued the unc-49 shrinker phenotype (FIG. 1) although complete rescuerequired the addition of 4 kb of 5′ flanking DNA (see Materials andMethods). We confirmed that the T21C12.1 open reading frame correspondedto the unc-49 gene by demonstrating that all unc-49 mutations werecontained within T21C12.1 (see below).

[0205] The structure of the unc-49 locus is very different from atypical ligand-gated ion channel subunit gene. At its 5′ end, unc-49contains a single region encoding the amino terminal half of a GABAreceptor subunit. The rest of the locus is made up of three repeatedregions, designated A, B and C, each encoding the carboxy-terminal halfof a subunit (FIG. 2A). The amino-terminal region encodes most of theextracellular residues, including two of the four loops thought to formthe ligand-binding site, and the absolutely-conserved disulfide-bondedloop. The three repeats encode the other two putative ligand-bindingloops, corresponding to the BDI and BDII GABA binding domains identifiedby Amin, J., and Weiss, D. S. (1994). Homomeric rho 1 GABA channels:activation properties and domains. Receptors and Channels 2, 227-236,and all four membrane-spanning domains.

[0206] The unc-49 Locus Encodes Three Distinct Subunits

[0207] We analyzed the structures of the mRNAs produced from unc-49 anddemonstrated that unc-49 is a compound locus which produces multiplereceptor subunits. Three full-length subunits, UNC-49A, UNC-49B andUNC-A9C, are generated by splicing the exons encoding the amino-terminalhalf of a subunit to the exons encoding the carboxy-terminal repeats A,B, and C, respectively (FIG. 2B). In addition, multiple isoforms ofUNC-49B and UNC-49C mRNAs were isolated. One UNC-49B isoform containsthe UNC-49C exons in its 3′ untranslated region. The other, UNC-49B′,appears to end within the intron immediately following the UNC-49B stopcodon. Alternative splicing within the UNC-49B coding region generatesthree additional isoforms (UNC-49B.1-3) which differ in theintracellular loop between M3 and M4 (See Materials and Methods). Twoisoforms of UNC-49C were isolated. One encodes a normal, full-lengthsubunit while the other, UNC-49Cshort, encodes an unusual subunittruncated at its amino terminus (FIG. 2B). Northern blot analysis ofmRNA isolated from C. elegans hermaphrodites confirmed that each of themajor classes of unc-49 mRNA is produced (FIG. 2C). Bands correspondingto the UNC-49B, UNC-49B′, UNC-49C, and UNC-49Cshort mRNA species wereeasily-detected. UNC-49A-specific bands were also detected, although atmuch lower levels. In addition, a number of large RNAs were identifiedwhich may represent splicing intermediates (see asterisks). Quantitativeanalysis of this Northern blot revealed that UNC-49B and UNC-49C mRNAare present at roughly equal levels, while the UNC-49Cshort mRNA istwo-fold less abundant, and the UNC-49A mRNA is 35-fold less abundant(see Materials and Methods).

[0208] The mRNA species encoding the UNC-49A, UNC-49B and UNC-49Csubunits share considerable structural overlap. The 5′ ends of thelongest respective cDNA clones are within 90 nucleotides of one another.Thus, transcription of all three full-length subunits initiates at aboutthe same place, suggesting that they are all under the control of thesame promoter. Moreover, the subunits encoded by these mRNA speciesshare a stretch of 188 identical amino terminal residues at their aminotermini (FIG. 3). However, the remaining parts, which contain most ofthe known determinants of subunit function, are encoded by differentsets of exons. UNC-49Cshort, by contrast, shares no sequence identitywith UNC-49A or UNC-49B. However, UNC-49Cshort overlaps completely withthe carboxy-terminal portion of repeat C, with the exception of fouramino acids at the UNC-49Cshort amino terminus (FIG. 3).

[0209] Structural Features of the GABA Receptor Subunits Encoded byunc-49

[0210] The existence of three full-length subunits within the unc-49locus led us to speculate that the UNC-49 subunits may functionallycorrespond to the α, β and γ subunits of vertebrate GABA_(A) receptors.To evaluate whether the UNC-49 subunits are closely-related to thevertebrate subunits, we performed phylogenetic comparisons using acomprehensive set of ligand-gated chloride channel subunits. Thisanalysis demonstrated that the UNC-49 subunits are not orthologous toany of the vertebrate GABA_(A) receptor subunit classes, but moreclosely resemble the Drosophila melanogaster rdl gene product (FIG. 4A).Because the UNC-49 proteins share a common amino terminus, they aregrouped into a closely-related family. To eliminate this bias from ouranalysis, we aligned only the carboxy-terminal segments of theligand-gated chloride channels. The results (not shown) were largely thesame as those using full-length subunits except that UNC-49C, which isvery divergent, forms a unique subunit class.

[0211] Sequence comparisons showed strong conservation between theUNC-49 subunits and other ligand-gated chloride channels, howeversequence differences are seen in the GABA binding domains and poreregions. Two putative GABA binding domains, BDI and BDII, have beendefined by structure-function studies of the vertebrate β and ρ GABAreceptor subunits. Within BDI, UNC-49A and UNC-49C contain a serineresidue, and UNC-49B contains a glutamic acid residue at a positionwhere the vertebrate β and ρ subunits and Drosophila rdl contain athreonine residue (marked with a # symbol in FIG. 4B). In BDII, allunc-49 subunits contain a serine residue at a position where the β, ρand rdl subunits contain a threonine residue (marked with a $ symbol inFIG. 5B). Mutating the threonine residues in BDI or in BDII to serine inthe β and ρ subunits results in significantly-decreased GABAresponsiveness. These differences suggest that the unc-49-encodedreceptor should be relatively insensitive to GABA, however thisprediction is not fully borne out in physiological studies (see below).In the pore domain, UNC-49A and UNC-49B show a high degree of sequenceconservation with the vertebrate GABA_(A) receptor subunits but UNC-49Cis highly divergent (FIG. 4B). One of the divergent residues in theUNC-49C pore domain is a glutamic acid residue (marked with @, FIG. 4B).Negatively-charged residues are generally not found in the pore domainsof ligand-gated chloride channels. One exception is the β subunit of thevertebrate glycine receptor. This residue causes reduced single channelconductance in heteromeric glycine receptors containing the β subunit,and physiological studies (see below) demonstrate that UNC-49C subunitsconfer similarly reduced single-channel conductance. Despite itsdivergent sequence, UNC-49C is not expected to display altered ionselectivity because residues within M2 known to affect ion selectivityare conserved.

[0212] Finally, the intracellular loops of the UNC-49 subunits containseveral potential protein kinase A, protein kinase C and casein kinaseII phosphorylation sites (FIG. 4C). Surprisingly, none of the consensusphosphorylation sites within the UNC-49B intracellular loop are affectedby the alternative splicing within this domain (FIG. 4C). This findingwas unexpected because the number of phosphorylation sites in thevertebrate β2 and γ2 GABA_(A) receptor subunits is regulated byalternative splicing.

[0213] All unc-49 Mutations Affect UNC-49B

[0214] Although unc-49 encodes multiple subunits, an analysis of unc-49mutations indicated that only mutations which affect UNC-49B eliminatereceptor function. First, inter se crosses determined that there is onlya single complementation group within the unc-49 locus. Second, allalleles disrupt the UNC-49B subunit. The unc-49(n1324) insertion (FIG.5A), and the point mutations in the e407 and n2392 alleles affect thecommon amino terminus shared by the three full-length subunits (FIG. 5B,C). By contrast, the unc-49(e929) polymorphism (FIG. 5A), and the pointmutations in the e382, e468 and e641 alleles disrupt UNC-49Bspecifically. These three alleles each contain a missense mutation whichsubstitutes a charged residue for the highly-conserved glycine residuefound in the BDI motif of UNC-49B (FIG. 5B, C). The presence of acharged residue in the mutant subunits probably disrupts secondarystructure within the ligand binding pocket because the glycine at thisposition is thought to be required for the formation of a hairpin turn.Third, we demonstrated that the UNC-49B open reading frame wassufficient for rescue. Specifically, rescuing fragments lacking UNC-49Aor UNC-49C were capable of rescuing unc-49(e382) while a rescuingfragment lacking UNC-49B was not (not shown). Thus, loss of UNC-49Balone is sufficient to cause the shrinker phenotype.

[0215] UNC-49B and UNC-49C are Co-localized at the NeuromuscularJunction.

[0216] Because of its operon-like structure, we hypothesized that unc-49may encode a heteromeric GABA receptor by producing multipleco-assembling GABA receptor subunits. This hypothesis predicts that theUNC-49 subunits will be co-expressed and co-localized withinpostsynaptic cells. We tested subunit co-localization by inserting thegreen fluorescent protein (GFP) into the large intracellular loop ofeach subunit in a plasmid encompassing the entire locus. The resultingconstructs, UNC-49A::GFP, UNC-49B::GFP and UNC-49C::GFP, each produceall of the UNC-49 subunits, one of which is tagged with GFP (A, B or C,respectively; FIG. 6A). These constructs were able to rescue theshrinker phenotype of unc-49(e382) mutants. In addition, we introduced astop codon into the common amino-terminal region of the UNC-49C::GFPconstruct to create an UNC-49Cshort::GFP construct (FIG. 6A). Thisconstruct did not encode any full-length UNC-49 subunits, and did notrescue the shrinker phenotype.

[0217] Using these constructs, we demonstrated that UNC-49B and UNC-49Care co-localized to the neuromuscular junction. The UNC-49A::GFP andUNC-49Cshort::GFP constructs did not produce detectable GFPfluorescence. Based on the low levels of UNC-49A mRNA detected byNorthern analysis, the lack of UNC-49A::GFP expression was notsurprising, but the lack of UNC-49Cshort fluorescence was unexpected. Weconclude that the UNC-49Cshort mRNA is not efficiently translated in C.elegans hermaphrodites. By contrast, transgenic worms carrying theUNC-49B::GFP and UNC-49C::GFP constructs produced very similar patternsof GFP fluorescence (FIGS. 6B, C). In both cases, fluorescence wasdetected mainly in the head and body wall muscles on both the dorsal andventral sides. Within these cells, the GFP fluorescence was brightestwhere the nerves and muscles make contact, suggesting that bothGFP-tagged subunits are efficiently localized to the neuromuscularjunctions. The only consistent difference between the two expressionpatterns was that strong GFP fluorescence was observed in the sphinctermuscle in UNC-49B::GFP animals but not in UNC-49C::GFP animals (rightpanel, FIGS. 6B, C). We conclude that the expression patterns ofUNC-49B::GFP and UNC-49C::GFP are consistent with the hypothesis thatthese two subunits function as a heteromeric GABA receptor.

[0218] UNC-49B and UNC-49C Physically Interact at the NeuromuscularJunction

[0219] As further evidence that UNC-49B and UNC-49C interact, wedemonstrated that the localization of UNC-49C to the neuromuscularjunction requires UNC-49B expression. We expressed GFP-tagged UNC-49B inthe absence of UNC-49C by inactivating the UNC-49C open reading frame inthe UNC-49B::GFP construct (FIG. 7A). This modified UNC-49B::GFPconstruct was injected into unc-49(e407) animals, which lack allfull-length unc-49 subunits because of a nonsense mutation in the commonamino-terminal region. The resulting transgenic lines showed punctatefluorescence at neuromuscular junctions (FIG. 7B) which wasindistinguishable from the pattern observed when UNC-49C was present. Wealso performed the reciprocal experiment: GFP-tagged UNC-49C wasexpressed in the absence of UNC-49B by inactivating the UNC-49B openreading frame in the UNC-49C::GFP construct. This construct was alsoinjected into unc-49(e407) animals. GFP fluorescence was clearly visibleon muscle membranes, but not at the neuromuscular junction (FIG. 7C,compare with FIG. 6D). Thus, synaptic localization of UNC-49C depends onthe presence of UNC-49B.

[0220] UNC-49B and UNC-49C Co-assemble in Heterologous Cells

[0221] We confirmed that UNC-49B and UNC-49C co-assemble to form aheteromultimer by demonstrating that co-expression of UNC-49B andUNC-49C in heterologous cells resulted in a functionally-distinct GABAreceptor. UNC-49B formed a homomeric GABA receptor in Xenopus.oocytes.Oocytes injected with UNC-49B.1 mRNA were analyzed using thetwo-electrode voltage clamp technique. These cells displayed a robust,desensitizing, dose-dependent current when exposed to GABA (FIG. 8A). Ina representative experiment, the GABA concentration required to producehalf-maximal channel activity (EC₅₀) was 43.7 μM, and the Hillcoefficient was 2.94, suggesting that a minimum of three GABA moleculesare required to open the channel (FIG. 8B, Table 1). The reversalpotential for this current was −30 mV (not shown), which is consistentwith a chloride conductance. UNC-49B receptors are highly GABAselective. UNC-49B-expressing oocytes did not respond to eitherglutamate or glycine applied at 1 mM or 10 mM. Applications of 10 mMβ-alanine produced currents that were only slightly greater thanbaseline noise (n=4, not shown). By contrast, UNC-49C was not able toform a homomeric GABA receptor. Xenopus oocytes injected with UNC-49CRNA failed to respond to GABA at any concentration, and were equallyunresponsive to glutamate, glycine and β-alanine. UNC-49A, andUNC-49Cshort were also unable to form homomeric GABA receptors. WhenUNC-49B and UNC-49C subunits were co-expressed, a functionally distinctreceptor was formed. Xenopus oocytes were injected with equal amounts ofUNC-49B and UNC-49C RNA. The EC₅₀ value for GABA on these oocytes was107.5 μM, and the Hill coefficient was 1.33 (FIG. 8B, Table 1). The GABAdose-response curves were accurately fit with a single Hill equation,which suggests that only a single population of receptors is present.This result argues UNC-49B and UNC-49C co-assemble very efficiently,such that the homomeric assembly of UNC-49B is eliminated or greatlyreduced.

[0222] We confirmed that the UNC-49C subunit co-assembles efficientlywith the UNC-49B subunit by co-expressing these subunits in HEK-293fibroblast cells and performing single channel recordings. In cellsexpressing UNC-49B alone, we observed a single main conductance state of37.5 pS. In cells transfected with the UNC-49B and UNC-49C, we observeda single main conductance state of 30.9 pS (FIG. 8C, Table 1). We didnot observe significant numbers of channel openings corresponding toUNC-49B homomers in cells expressing both UNC-49B and UNC-49C. Althoughabout 10% of channel openings in these cells were larger than the 30.9pS main conductance, their conductance was roughly twice as large as themain conductance, suggesting that they corresponded to two UNC-49B/Cheteromeric channels opening simultaneously. Thus in HEK-293 cells, likein Xenopus oocytes, UNC-49B and UNC-49C co-assemble, and the presence ofUNC-49C effectively suppresses the homomeric assembly of UNC-49B,suggesting that co-assembly is efficient.

EXAMPLE 3. Discussion

[0223] We have cloned the C. elegans unc-49 gene and demonstrated thatit is a compound locus encoding three GABA receptor subunits, UNC-49A,UNC-49B and UNC-49C, by splicing a common amino terminus to one of threealternative carboxy termini. Complex loci in C. elegans often produceproteins which function within the same genetic pathway. A simplehypothesis for how the UNC-49 subunits could function together is thatthey form a heteromeric GABA receptor.

[0224] To test this hypothesis, we first determined whether the UNC-49subunits were co-localized. The UNC-49A and the UNC-49Cshort isoformwere not expressed at significant levels in adult hermaphrodites.However, we demonstrated that UNC-49B and UNC-49C were both expressed athigh levels in muscle cells, and were localized to the neuromuscularjunctions. Furthermore, we demonstrated an interaction between theUNC-49B and UNC-49C subunits by showing that the synaptic localizationof UNC-49C required the presence of an intact UNC-49B subunit. Thesimplest interpretation of this result is that UNC-49B binds to aprotein required for proper receptor localization, such as a receptortethering protein, and UNC-49C binds to UNC-49B (FIG. 9). These dataprovide the first direct evidence for the physical interaction of twoligand-gated ion channel subunits at an intact synapse. Previous studieshave shown that ligand-gated ion channel subunits, and specifically GABAreceptor subunits can be co-immunoprecipitated from brain extracts,however these results do not demonstrate that subunits are physicallyassociated at synapses. As a final test of the hypothesis, wedemonstrated that UNC-49B and UNC-49C form heteromeric GABA receptorswhen co-expressed in heterologous cells. Moreover, this co-assemblyappeared to be very efficient because the formation of UNC-49B/Cheteromers was strongly preferred over the assembly of UNC-49B homomerseven though UNC-49B homomer formation occurred readily in the absence ofUNC-49C. Taken together, our data make a compelling argument thatUNC-49B co-assembles with UNC-49C to form a heteromeric GABA receptor atthe C. elegans neuromuscular junction.

[0225] These results establish a novel mechanism to regulate theco-expression of subunits which assemble into heteromeric ion channels.In vertebrates, heteromers are assembled from subunits encoded byseparate genes. The promoters of these genes share functionalsimilarities, resulting in overlapping subunit expression patterns. Bycontrast, unc-49 achieves coordinate regulation of co-assembled subunitsby driving their expression from a single promoter. Regulation may alsooccur at the level of mRNA splicing because the UNC-49A, UNC-49B andUNC-49C expression patterns are not identical. Thus, the unc-49 geneorganization could permit the production of several different GABAreceptor isoforms. A similar gene organization has been observed forother subunit genes in C. elegans, and the related nematode Haemonchtiscontortis. Thus nematodes in general may encode heteromeric ligand-gatedion channels using this type of compound locus. The differences ingenomic organization between vertebrates and nematodes suggest thatheteromultimeric receptor structure may not share a common evolutionaryantecedent, but instead arose independently in the two lineages. Insupport of this view, the UNC-49 subunits are clearly not orthologous tothe GABA_(A) receptor subunits, which are known to form heteromericreceptors.

[0226] The addition of UNC-49C subunits to the UNC-49B GABA receptorresulted in reduced GABA sensitivity, reduced chloride conductance, andreduced cooperativity of channel gating. Examination of the UNC-49Csequence suggests a structural basis for some of its modulatoryfunctions. Reduced GABA sensitivity may be caused by a serine residuewithin the BDI domain of UNC-49C. Typically, GABA receptor subunitscontain a threonine residue at this position. Substitution of serine forthis threonine in vertebrate β and ρ GABA receptor subunits has beenshown to greatly reduce GABA sensitivity. Thus, UNC-49C is anaturally-occurring subunit variant which confirms the importance of theBDI sequence for efficient channel gating by GABA as suggested bysite-directed mutagenesis experiments. Reduced chloride conductanceprobably results from the presence of a negatively-charged glutamic acidresidue within the pore-lining M2 domain. Of the 35 ligand-gatedchloride channels examined, only UNC-49C and the glycine receptor βsubunit contain a negatively-charged residue within M2. This glutamicacid residue in the glycine receptor β subunit was demonstrated to causereduced single channel conductance in glycine α/β heteromers compared toglycine αhomomers. Thus, the glutamic acid residue in UNC-49C is likelyto cause the reduced single channel conductance in UNC-49B/C heteromerscompared to UNC-49B homomers, presumably by acting as an electrostaticimpediment to the passage of negatively-charged chloride ions throughthe channel pore.

[0227] This study of unc-49 represents a complete vertical analysis of aneurotransmitter receptor. Analysis of a mutant lacking GABAneurotransmission led to the isolation of the unc-49 gene. Molecularcharacterization of unc-49 demonstrated that it encodes multiple GABAreceptor subunits in an operon-like arrangement. Expression studies invivo demonstrated that two of the subunits are co-localized toneuromuscular junctions where they co-assemble to form a heteromericreceptor. Finally, electrophysiological studies confirmed that theheteromeric receptor functions as a GABA-gated chloride channel, anddemonstrated that one subunit is absolutely required for receptorfunction, while the other plays a modulatory role. Moreover, a specificstructural basis for the function of the modulatory subunit wassuggested by the sequence of its GABA-binding and pore-lining domains.The demonstration that GFP-tagged UNC-49 subunits are properlyco-assembled into receptors and localized to synapses is of particularsignificance for future studies. Mechanisms for the assembly andlocalization of ligand-gated ion channels have been difficult to studyin intact nervous systems. C. elegans offers a genetic approach to thestudy of these processes in the context of the functioning synapse.

1 18 1 1652 PRT Caenorhabditis elegans 1 Cys Gly Gly Ala Ala Ala Ala AlaCys Cys Thr Cys Cys Cys Cys Ala 1 5 10 15 Ala Cys Ala Thr Thr Gly GlyCys Thr Cys Ala Cys Ala Cys Cys Cys 20 25 30 Gly Gly Ala Thr Thr Ala ThrGly Ala Thr Cys Thr Thr Cys Thr Gly 35 40 45 Cys Thr Gly Cys Thr Cys CysThr Gly Cys Thr Gly Cys Thr Cys Cys 50 55 60 Thr Thr Cys Thr Gly Cys ThrGly Thr Ala Gly Thr Thr Gly Ala Gly 65 70 75 80 Ala Cys Gly Ala Ala GlyAla Ala Gly Ala Ala Gly Ala Ala Gly Ala 85 90 95 Ala Gly Cys Thr Cys CysAla Thr Thr Cys Thr Cys Gly Ala Gly Ala 100 105 110 Ala Ala Thr Gly GlyCys Thr Cys Gly Thr Cys Cys Ala Thr Thr Cys 115 120 125 Ala Cys Ala CysThr Thr Ala Thr Cys Gly Thr Ala Cys Thr Cys Cys 130 135 140 Thr Cys ThrCys Cys Gly Cys Ala Cys Ala Thr Cys Thr Gly Thr Gly 145 150 155 160 ThrCys Thr Ala Cys Ala Thr Gly Thr Gly Gly Thr Thr Gly Thr Gly 165 170 175Ala Cys Ala Cys Ala Gly Gly Ala Thr Gly Ala Gly Gly Ala Cys Thr 180 185190 Cys Ala Cys Ala Thr Ala Thr Cys Ala Ala Cys Ala Cys Thr Cys Ala 195200 205 Ala Cys Thr Cys Cys Thr Cys Thr Cys Ala Thr Cys Ala Gly Thr Thr210 215 220 Cys Thr Cys Gly Ala Thr Ala Gly Ala Cys Thr Cys Ala Cys GlyAla 225 230 235 240 Ala Thr Cys Gly Cys Ala Cys Thr Ala Cys Thr Thr AlaThr Gly Ala 245 250 255 Thr Ala Ala Ala Ala Gly Ala Thr Thr Ala Cys GlyGly Cys Cys Cys 260 265 270 Ala Gly Gly Thr Ala Thr Gly Gly Thr Gly AlaAla Ala Ala Gly Cys 275 280 285 Cys Ala Gly Thr Cys Gly Ala Cys Gly ThrThr Gly Gly Ala Ala Thr 290 295 300 Thr Ala Cys Gly Ala Thr Ala Cys AlaCys Gly Thr Thr Thr Cys Thr 305 310 315 320 Thr Cys Ala Ala Thr Cys ThrCys Thr Gly Cys Ala Gly Thr Thr Thr 325 330 335 Cys Ala Gly Ala Ala GlyThr Thr Gly Ala Thr Ala Thr Gly Gly Ala 340 345 350 Cys Thr Thr Cys AlaCys Ala Thr Thr Ala Gly Ala Cys Thr Thr Cys 355 360 365 Thr Ala Cys AlaThr Gly Cys Gly Thr Cys Ala Ala Ala Cys Gly Thr 370 375 380 Gly Gly CysAla Ala Gly Ala Cys Cys Cys Thr Cys Gly Ala Cys Thr 385 390 395 400 AlaGly Cys Cys Thr Thr Cys Gly Gly Ala Ala Gly Thr Cys Thr Thr 405 410 415Gly Ala Thr Thr Thr Gly Gly Gly Ala Cys Thr Thr Thr Cys Cys Ala 420 425430 Ala Ala Gly Ala Ala Ala Thr Cys Gly Ala Cys Thr Cys Ala Cys Thr 435440 445 Thr Ala Cys Cys Gly Thr Cys Gly Gly Ala Gly Thr Ala Gly Ala Cys450 455 460 Thr Ala Cys Cys Thr Gly Gly Ala Thr Ala Gly Ala Cys Thr GlyThr 465 470 475 480 Gly Gly Ala Ala Ala Cys Cys Cys Gly Ala Cys Ala CysGly Thr Thr 485 490 495 Cys Thr Thr Cys Cys Cys Ala Ala Ala Thr Gly AlaAla Ala Ala Gly 500 505 510 Ala Ala Ala Thr Cys Ala Thr Thr Cys Thr ThrCys Cys Ala Cys Thr 515 520 525 Thr Gly Gly Cys Ala Ala Cys Cys Ala CysAla Cys Ala Thr Ala Ala 530 535 540 Cys Thr Cys Gly Thr Thr Cys Cys ThrThr Cys Gly Thr Ala Thr Cys 545 550 555 560 Gly Ala Gly Gly Gly Thr GlyAla Thr Gly Gly Ala Ala Cys Gly Gly 565 570 575 Thr Thr Thr Ala Thr AlaCys Thr Ala Gly Thr Cys Ala Ala Ala Gly 580 585 590 Ala Thr Thr Ala AlaCys Ala Gly Thr Cys Ala Cys Thr Gly Cys Ala 595 600 605 Ala Cys Gly ThrGly Thr Cys Cys Ala Ala Thr Gly Gly Ala Cys Cys 610 615 620 Thr Gly AlaAla Gly Cys Thr Gly Thr Thr Cys Cys Cys Ala Ala Thr 625 630 635 640 GlyGly Ala Cys Thr Cys Thr Cys Ala Ala Cys Ala Cys Thr Gly Thr 645 650 655Ala Ala Ala Cys Thr Gly Gly Ala Ala Ala Thr Thr Gly Ala Ala Ala 660 665670 Gly Cys Thr Ala Thr Gly Cys Gly Thr Ala Thr Thr Cys Gly Ala Cys 675680 685 Gly Gly Cys Cys Gly Ala Ala Ala Thr Cys Gly Ala Gly Thr Ala Cys690 695 700 Ala Ala Ala Thr Gly Gly Thr Gly Thr Ala Cys Gly Thr Cys GlyAla 705 710 715 720 Ala Gly Gly Ala Gly Cys Cys Gly Ala Ala Thr Thr GlyThr Thr Cys 725 730 735 Gly Ala Cys Ala Gly Cys Gly Gly Thr Cys Ala AlaGly Gly Cys Cys 740 745 750 Gly Ala Cys Gly Cys Gly Ala Ala Cys Ala ThrCys Gly Ala Ala Cys 755 760 765 Thr Gly Thr Cys Gly Ala Gly Thr Thr AlaThr Ala Ala Ala Thr Thr 770 775 780 Cys Ala Cys Thr Ala Ala Ala Ala ThrCys Thr Gly Cys Cys Ala Ala 785 790 795 800 Ala Ala Ala Cys Gly Gly AlaCys Ala Cys Thr Thr Gly Cys Cys Ala 805 810 815 Gly Cys Ala Cys Thr ThrCys Ala Thr Cys Gly Gly Gly Gly Ala Cys 820 825 830 Cys Thr Ala Cys ThrCys Thr Cys Gly Thr Cys Thr Ala Cys Gly Gly 835 840 845 Gly Thr Thr AlaGly Thr Thr Thr Cys Ala Thr Ala Thr Thr Thr Gly 850 855 860 Ala Thr CysGly Cys Gly Ala Cys Ala Gly Cys Gly Gly Cys Thr Thr 865 870 875 880 CysThr Ala Cys Thr Thr Thr Cys Thr Thr Cys Ala Ala Ala Thr Ala 885 890 895Thr Thr Thr Thr Thr Cys Cys Cys Thr Gly Cys Cys Ala Gly Cys Cys 900 905910 Thr Cys Gly Thr Cys Gly Thr Ala Gly Thr Thr Thr Thr Ala Thr Cys 915920 925 Ala Thr Gly Gly Ala Thr Cys Thr Cys Ala Thr Thr Cys Thr Gly Gly930 935 940 Ala Thr Cys Ala Ala Thr Cys Gly Thr Gly Ala Cys Thr Cys GlyGly 945 950 955 960 Cys Gly Cys Cys Thr Thr Cys Gly Cys Gly Ala Ala CysCys Cys Thr 965 970 975 Ala Ala Thr Cys Gly Gly Thr Ala Cys Gly Ala ThrGly Ala Cys Gly 980 985 990 Gly Thr Gly Cys Thr Cys Ala Cys Thr Gly AlaGly Ala Cys Thr Cys 995 1000 1005 Ala Thr Cys Thr Thr Ala Thr Gly AlaCys Cys Gly Gly Ala Ala Cys 1010 1015 1020 Cys Ala Ala Thr Cys Gly AlaCys Gly Thr Cys Thr Thr Cys Cys Ala 1025 1030 1035 1040 Cys Cys Ala GlyThr Thr Gly Cys Cys Thr Ala Thr Gly Thr Ala Ala 1045 1050 1055 Ala AlaGly Cys Cys Gly Thr Thr Gly Ala Thr Gly Thr Ala Thr Thr 1060 1065 1070Cys Cys Thr Cys Gly Gly Thr Thr Thr Cys Thr Gly Cys Thr Ala Thr 10751080 1085 Cys Thr Thr Cys Thr Gly Gly Thr Thr Ala Thr Ala Cys Thr GlyGly 1090 1095 1100 Cys Gly Thr Thr Gly Ala Thr Cys Gly Ala Gly Thr AlaCys Gly Cys 1105 1110 1115 1120 Cys Thr Gly Thr Gly Thr Thr Gly Cys CysThr Ala Cys Thr Cys Ala 1125 1130 1135 Ala Ala Ala Ala Ala Gly Ala AlaGly Ala Ala Cys Gly Ala Gly Gly 1140 1145 1150 Ala Thr Cys Gly Thr CysGly Gly Ala Gly Ala Ala Gly Ala Gly Ala 1155 1160 1165 Gly Ala Ala GlyAla Ala Gly Ala Cys Gly Gly Ala Gly Cys Ala Thr 1170 1175 1180 Ala AlaAla Cys Cys Thr Gly Cys Thr Cys Cys Gly Cys Cys Gly Ala 1185 1190 11951200 Cys Ala Cys Cys Thr Gly Ala Thr Ala Thr Thr Cys Thr Thr Cys Ala1205 1210 1215 Cys Gly Ala Cys Gly Thr Cys Cys Gly Cys Cys Thr Thr GlyCys Cys 1220 1225 1230 Gly Ala Ala Thr Gly Cys Ala Cys Ala Thr Gly CysAla Ala Cys Gly 1235 1240 1245 Cys Gly Gly Cys Thr Cys Cys Ala Ala CysCys Thr Cys Gly Ala Thr 1250 1255 1260 Cys Ala Thr Cys Gly Cys Cys GlyThr Cys Ala Thr Cys Ala Ala Gly 1265 1270 1275 1280 Cys Ala Gly Thr CysGly Ala Ala Thr Cys Gly Ala Thr Thr Cys Thr 1285 1290 1295 Gly Thr GlyThr Cys Ala Gly Thr Cys Ala Cys Ala Gly Thr Cys Ala 1300 1305 1310 CysAla Thr Thr Gly Ala Cys Ala Thr Cys Gly Thr Cys Ala Gly Cys 1315 13201325 Cys Gly Thr Gly Cys Cys Gly Cys Gly Thr Thr Thr Cys Cys Thr Cys1330 1335 1340 Thr Thr Gly Thr Thr Thr Thr Cys Ala Thr Cys Thr Thr GlyThr Thr 1345 1350 1355 1360 Cys Ala Ala Cys Ala Cys Thr Cys Thr Cys ThrThr Cys Thr Gly Gly 1365 1370 1375 Cys Thr Gly Ala Thr Thr Cys Thr AlaCys Thr Gly Thr Ala Cys Ala 1380 1385 1390 Ala Ala Thr Cys Cys Ala AlaGly Cys Gly Thr Cys Thr Gly Cys Cys 1395 1400 1405 Gly Thr Ala Thr AlaThr Thr Ala Gly Thr Gly Ala Ala Cys Ala Cys 1410 1415 1420 Gly Ala GlyGly Gly Thr Gly Ala Cys Cys Gly Thr Thr Gly Cys Gly 1425 1430 1435 1440Ala Thr Gly Cys Thr Cys Cys Ala Gly Ala Cys Cys Thr Thr Cys Ala 14451450 1455 Thr Thr Ala Ala Thr Cys Thr Cys Ala Ala Thr Cys Cys Ala AlaCys 1460 1465 1470 Thr Thr Cys Cys Thr Cys Ala Thr Cys Ala Thr Thr ThrThr Cys Cys 1475 1480 1485 Ala Thr Thr Thr Cys Gly Ala Ala Thr Ala ThrCys Thr Cys Thr Thr 1490 1495 1500 Thr Thr Thr Cys Thr Thr Gly Cys AlaCys Ala Gly Ala Ala Gly Cys 1505 1510 1515 1520 Cys Thr Thr Thr Thr ThrThr Cys Gly Thr Thr Thr Thr Thr Thr Thr 1525 1530 1535 Thr Thr Ala ThrThr Gly Ala Thr Thr Thr Ala Thr Thr Thr Thr Thr 1540 1545 1550 Ala CysGly Gly Ala Thr Thr Thr Thr Thr Ala Gly Ala Thr Ala Ala 1555 1560 1565Thr Gly Cys Ala Cys Ala Gly Ala Thr Gly Cys Cys Thr Cys Ala Thr 15701575 1580 Thr Gly Cys Thr Cys Ala Ala Ala Thr Ala Ala Ala Thr Thr ThrAla 1585 1590 1595 1600 Thr Thr Thr Thr Ala Ala Thr Thr Gly Thr Cys GlyAla Ala Ala Ala 1605 1610 1615 Ala Ala Ala Ala Ala Ala Ala Ala Ala AlaAla Ala Ala Ala Ala Ala 1620 1625 1630 Ala Ala Ala Ala Ala Ala Ala AlaAla Ala Ala Ala Ala Ala Ala Ala 1635 1640 1645 Ala Ala Ala Ala 1650 21652 DNA Caenorhabditis elegans 2 cggaaaaacc tccccaacat tggctcacacccggattatg atcttctgct gctcctgctg 60 ctccttctgc tgtagttgag acgaagaagaagaagaagct ccattctcga gaaatggctc 120 gtccattcac acttatcgta ctcctctccgcacatctgtg tctacatgtg gttgtgacac 180 aggatgagga ctcacatatc aacactcaactcctctcatc agttctcgat agactcacga 240 atcgcactac ttatgataaa agattacggcccaggtatgg tgaaaagcca gtcgacgttg 300 gaattacgat acacgtttct tcaatctctgcagtttcaga agttgatatg gacttcacat 360 tagacttcta catgcgtcaa acgtggcaagaccctcgact agccttcgga agtcttgatt 420 tgggactttc caaagaaatc gactcacttaccgtcggagt agactacctg gatagactgt 480 ggaaacccga cacgttcttc ccaaatgaaaagaaatcatt cttccacttg gcaaccacac 540 ataactcgtt ccttcgtatc gagggtgatggaacggttta tactagtcaa agattaacag 600 tcactgcaac gtgtccaatg gacctgaagctgttcccaat ggactctcaa cactgtaaac 660 tggaaattga aagctatgcg tattcgacggccgaaatcga gtacaaatgg tgtacgtcga 720 aggagccgaa ttgttcgaca gcggtcaaggccgacgcgaa catcgaactg tcgagttata 780 aattcactaa aatctgccaa aaacggacacttgccagcac ttcatcgggg acctactctc 840 gtctacgggt tagtttcata tttgatcgcgacagcggctt ctactttctt caaatatttt 900 tccctgccag cctcgtcgta gttttatcatggatctcatt ctggatcaat cgtgactcgg 960 cgccttcgcg aaccctaatc ggtacgatgacggtgctcac tgagactcat cttatgaccg 1020 gaaccaatcg acgtcttcca ccagttgcctatgtaaaagc cgttgatgta ttcctcggtt 1080 tctgctatct tctggttata ctggcgttgatcgagtacgc ctgtgttgcc tactcaaaaa 1140 agaagaacga ggatcgtcgg agaagagagaagaagacgga gcataaacct gctccgccga 1200 cacctgatat tcttcacgac gtccgccttgccgaatgcac atgcaacgcg gctccaacct 1260 cgatcatcgc cgtcatcaag cagtcgaatcgattctgtgt cagtcacagt cacattgaca 1320 tcgtcagccg tgccgcgttt cctcttgttttcatcttgtt caacactctc ttctggctga 1380 ttctactgta caaatccaag cgtctgccgtatattagtga acacgagggt gaccgttgcg 1440 atgctccaga ccttcattaa tctcaatccaacttcctcat cattttccat ttcgaatatc 1500 tctttttctt gcacagaagc cttttttcgtttttttttat tgatttattt ttacggattt 1560 ttagataatg cacagatgcc tcattgctcaaataaattta ttttaattgt cgaaaaaaaa 1620 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaa 1652 3 2544 PRT Caenorhabditis elegans 3 Ala Ala Gly Thr Thr Thr GlyAla Gly Ala Gly Thr Gly Ala Thr Ala 1 5 10 15 Thr Ala Gly Gly Ala GlyAla Ala Ala Ala Ala Cys Cys Thr Cys Cys 20 25 30 Cys Cys Ala Ala Cys AlaThr Thr Gly Gly Cys Thr Cys Ala Cys Ala 35 40 45 Cys Cys Cys Gly Gly AlaThr Thr Ala Thr Gly Ala Thr Cys Thr Thr 50 55 60 Cys Thr Gly Cys Thr GlyCys Thr Cys Cys Thr Gly Cys Thr Gly Cys 65 70 75 80 Thr Cys Cys Thr ThrCys Thr Gly Cys Thr Gly Thr Ala Gly Thr Thr 85 90 95 Gly Ala Gly Ala CysGly Ala Ala Gly Ala Ala Gly Ala Ala Gly Ala 100 105 110 Ala Gly Ala AlaGly Cys Thr Cys Cys Ala Thr Thr Cys Thr Cys Gly 115 120 125 Ala Gly AlaAla Ala Thr Gly Gly Cys Thr Cys Gly Thr Cys Cys Ala 130 135 140 Thr ThrCys Ala Cys Ala Cys Thr Thr Ala Thr Cys Gly Thr Ala Cys 145 150 155 160Thr Cys Cys Thr Cys Thr Cys Cys Gly Cys Ala Cys Ala Thr Cys Thr 165 170175 Gly Thr Gly Thr Cys Thr Ala Cys Ala Thr Gly Thr Gly Gly Thr Thr 180185 190 Gly Thr Gly Ala Cys Ala Cys Ala Gly Gly Ala Thr Gly Ala Gly Gly195 200 205 Ala Cys Thr Cys Ala Cys Ala Thr Ala Thr Cys Ala Ala Cys AlaCys 210 215 220 Thr Cys Ala Ala Cys Thr Cys Cys Thr Cys Thr Cys Ala ThrCys Ala 225 230 235 240 Gly Thr Thr Cys Thr Cys Gly Ala Thr Ala Gly AlaCys Thr Cys Ala 245 250 255 Cys Gly Ala Ala Thr Cys Gly Cys Ala Cys ThrAla Cys Thr Thr Ala 260 265 270 Thr Gly Ala Thr Ala Ala Ala Ala Gly AlaThr Thr Ala Cys Gly Gly 275 280 285 Cys Cys Cys Ala Gly Gly Thr Ala ThrGly Gly Thr Gly Ala Ala Ala 290 295 300 Ala Gly Cys Cys Ala Gly Thr CysGly Ala Cys Gly Thr Thr Gly Gly 305 310 315 320 Ala Ala Thr Thr Ala CysGly Ala Thr Ala Cys Ala Cys Gly Thr Thr 325 330 335 Thr Cys Thr Thr CysAla Ala Thr Cys Thr Cys Thr Gly Cys Ala Gly 340 345 350 Thr Thr Thr CysAla Gly Ala Ala Gly Thr Thr Gly Ala Thr Ala Thr 355 360 365 Gly Gly AlaCys Thr Thr Cys Ala Cys Ala Thr Thr Ala Gly Ala Cys 370 375 380 Thr ThrCys Thr Ala Cys Ala Thr Gly Cys Gly Thr Cys Ala Ala Ala 385 390 395 400Cys Gly Thr Gly Gly Cys Ala Ala Gly Ala Cys Cys Cys Thr Cys Gly 405 410415 Ala Cys Thr Ala Gly Cys Cys Thr Thr Cys Gly Gly Ala Ala Gly Thr 420425 430 Cys Thr Thr Gly Ala Thr Thr Thr Gly Gly Gly Ala Cys Thr Thr Thr435 440 445 Cys Cys Ala Ala Ala Gly Ala Ala Ala Thr Cys Gly Ala Cys ThrCys 450 455 460 Ala Cys Thr Thr Ala Cys Cys Gly Thr Cys Gly Gly Ala GlyThr Ala 465 470 475 480 Gly Ala Cys Thr Ala Cys Cys Thr Gly Gly Ala ThrAla Gly Ala Cys 485 490 495 Thr Gly Thr Gly Gly Ala Ala Ala Cys Cys CysGly Ala Cys Ala Cys 500 505 510 Gly Thr Thr Cys Thr Thr Cys Cys Cys AlaAla Ala Thr Gly Ala Ala 515 520 525 Ala Ala Gly Ala Ala Ala Thr Cys AlaThr Thr Cys Thr Thr Cys Cys 530 535 540 Ala Cys Thr Thr Gly Gly Cys AlaAla Cys Cys Ala Cys Ala Cys Ala 545 550 555 560 Thr Ala Ala Cys Thr CysGly Thr Thr Cys Cys Thr Thr Cys Gly Thr 565 570 575 Ala Thr Cys Gly AlaGly Gly Gly Thr Gly Ala Thr Gly Gly Ala Ala 580 585 590 Cys Gly Gly ThrThr Thr Ala Thr Ala Cys Thr Ala Gly Thr Cys Ala 595 600 605 Ala Ala GlyAla Thr Thr Ala Ala Cys Ala Gly Thr Cys Ala Cys Thr 610 615 620 Gly CysAla Ala Cys Gly Thr Gly Thr Cys Cys Ala Ala Thr Gly Gly 625 630 635 640Ala Cys Cys Thr Gly Ala Ala Gly Cys Thr Gly Thr Thr Cys Cys Cys 645 650655 Ala Ala Thr Gly Gly Ala Cys Thr Cys Thr Cys Ala Ala Cys Ala Cys 660665 670 Thr Gly Thr Ala Ala Ala Cys Thr Gly Gly Ala Ala Ala Thr Thr Gly675 680 685 Ala Ala Ala Gly Cys Thr Ala Cys Gly Gly Gly Thr Ala Cys GlyAla 690 695 700 Gly Ala Cys Gly Ala Ala Ala Gly Ala Thr Ala Thr Cys GlyAla Cys 705 710 715 720 Thr Ala Cys Thr Ala Thr Thr Gly Gly Gly Gly GlyAla Ala Gly Ala 725 730 735 Ala Gly Cys Gly Gly Ala Cys Thr Gly Ala ThrThr Thr Gly Gly Ala 740 745 750 Gly Ala Thr Ala Ala Cys Gly Gly Cys ThrGly Thr Cys Ala Ala Gly 755 760 765 Thr Thr Thr Gly Ala Thr Ala Cys CysThr Thr Cys Cys Ala Gly Thr 770 775 780 Thr Gly Cys Cys Gly Cys Ala GlyThr Thr Thr Cys Ala Gly Cys Cys 785 790 795 800 Ala Ala Cys Gly Cys ThrGly Thr Ala Thr Thr Thr Thr Gly Thr Gly 805 810 815 Ala Ala Thr Ala CysAla Ala Cys Thr Ala Ala Ala Gly Cys Cys Gly 820 825 830 Ala Gly Ala CysCys Thr Cys Ala Thr Cys Ala Gly Gly Ala Ala Ala 835 840 845 Ala Thr AlaCys Gly Thr Ala Cys Gly Cys Cys Thr Gly Gly Cys Gly 850 855 860 Cys ThrGly Gly Ala Ala Gly Thr Ala Ala Thr Ala Thr Thr Gly Gly 865 870 875 880Thr Thr Cys Gly Ala Ala Ala Thr Ala Thr Gly Gly Gly Cys Thr Thr 885 890895 Cys Thr Ala Cys Ala Cys Thr Ala Thr Gly Ala Ala Cys Ala Thr Cys 900905 910 Gly Thr Cys Ala Thr Cys Cys Cys Ala Thr Cys Cys Ala Thr Cys Cys915 920 925 Thr Gly Ala Thr Cys Gly Thr Cys Ala Cys Cys Ala Thr Ala ThrCys 930 935 940 Thr Thr Gly Gly Gly Thr Ala Thr Cys Ala Thr Thr Thr ThrGly Gly 945 950 955 960 Thr Thr Gly Ala Ala Thr Cys Gly Ala Gly Ala AlaGly Cys Thr Thr 965 970 975 Cys Gly Cys Cys Gly Gly Cys Thr Cys Gly AlaGly Thr Thr Gly Gly 980 985 990 Ala Thr Thr Gly Gly Gly Thr Gly Thr GlyAla Cys Thr Ala Cys Thr 995 1000 1005 Gly Thr Gly Cys Thr Cys Ala CysAla Ala Thr Gly Ala Cys Ala Ala 1010 1015 1020 Cys Thr Cys Thr Gly AlaThr Cys Ala Cys Thr Ala Cys Ala Ala Cys 1025 1030 1035 1040 Cys Ala AlaThr Ala Ala Thr Thr Cys Gly Ala Thr Gly Cys Cys Ala 1045 1050 1055 AlaAla Ala Gly Thr Gly Thr Cys Thr Thr Ala Thr Gly Thr Cys Ala 1060 10651070 Ala Gly Gly Gly Thr Cys Thr Gly Gly Ala Thr Gly Thr Gly Thr Thr1075 1080 1085 Thr Cys Thr Thr Ala Ala Thr Thr Thr Thr Thr Gly Thr ThrThr Cys 1090 1095 1100 Gly Thr Ala Ala Thr Gly Gly Thr Ala Thr Thr CysGly Cys Cys Thr 1105 1110 1115 1120 Cys Gly Thr Thr Gly Cys Thr Cys GlyAla Gly Thr Ala Cys Gly Cys 1125 1130 1135 Cys Ala Thr Ala Gly Thr AlaThr Cys Cys Thr Ala Cys Ala Thr Gly 1140 1145 1150 Ala Ala Thr Ala AlaAla Cys Gly Ala Cys Thr Gly Gly Thr Cys Cys 1155 1160 1165 Thr Cys CysGly Ala Cys Gly Gly Gly Ala Ala Ala Ala Ala Cys Gly 1170 1175 1180 AlaAla Gly Ala Ala Ala Ala Gly Cys Cys Gly Cys Cys Gly Ala Ala 1185 11901195 1200 Cys Ala Ala Cys Ala Gly Cys Ala Gly Cys Gly Ala Ala Ala CysGly 1205 1210 1215 Ala Gly Ala Thr Gly Cys Cys Ala Ala Thr Gly Thr ThrCys Ala Ala 1220 1225 1230 Cys Gly Cys Gly Ala Gly Cys Cys Cys Gly AlaAla Gly Gly Cys Cys 1235 1240 1245 Gly Cys Cys Ala Ala Thr Ala Ala ThrAla Ala Thr Gly Cys Thr Gly 1250 1255 1260 Ala Cys Thr Thr Gly Thr AlaCys Thr Thr Thr Gly Cys Cys Gly Gly 1265 1270 1275 1280 Ala Cys Ala CysAla Ala Thr Thr Cys Cys Thr Cys Thr Ala Thr Gly 1285 1290 1295 Ala AlaThr Cys Cys Ala Thr Thr Gly Ala Thr Gly Gly Ala Gly Ala 1300 1305 1310Thr Cys Cys Cys Ala Gly Ala Ala Ala Ala Thr Thr Gly Thr Gly Ala 13151320 1325 Thr Thr Gly Cys Cys Gly Gly Ala Cys Gly Ala Thr Thr Cys CysAla 1330 1335 1340 Ala Thr Gly Ala Thr Gly Cys Ala Ala Cys Ala Thr CysCys Ala Cys 1345 1350 1355 1360 Gly Thr Cys Thr Thr Gly Thr Cys Ala CysAla Gly Ala Cys Gly Gly 1365 1370 1375 Cys Gly Cys Ala Cys Ala Thr AlaCys Gly Cys Thr Ala Thr Gly Gly 1380 1385 1390 Cys Cys Gly Gly Cys ThrCys Cys Ala Thr Thr Cys Gly Cys Gly Cys 1395 1400 1405 Gly Gly Cys CysGly Ala Ala Ala Ala Ala Gly Gly Cys Thr Thr Cys 1410 1415 1420 Cys AlaAla Gly Ala Cys Ala Thr Gly Cys Thr Gly Cys Cys Ala Ala 1425 1430 14351440 Cys Gly Ala Thr Gly Gly Ala Cys Gly Cys Cys Thr Gly Cys Ala Ala1445 1450 1455 Ala Ala Ala Thr Cys Gly Ala Thr Ala Ala Gly Cys Thr ThrAla Gly 1460 1465 1470 Cys Cys Gly Ala Thr Ala Cys Gly Gly Thr Thr ThrCys Cys Cys Ala 1475 1480 1485 Thr Thr Gly Thr Cys Thr Thr Thr Cys ThrCys Thr Ala Thr Cys Thr 1490 1495 1500 Thr Cys Ala Ala Thr Ala Thr AlaGly Thr Cys Thr Ala Cys Thr Gly 1505 1510 1515 1520 Gly Thr Thr Gly ThrAla Thr Ala Thr Gly Ala Ala Ala Thr Ala Thr 1525 1530 1535 Cys Thr AlaAla Gly Cys Thr Thr Ala Ala Ala Cys Thr Cys Gly Thr 1540 1545 1550 CysGly Gly Ala Cys Ala Ala Gly Ala Thr Cys Cys Ala Gly Gly Ala 1555 15601565 Gly Ala Ala Cys Gly Ala Cys Ala Ala Gly Thr Gly Gly Cys Ala Gly1570 1575 1580 Cys Ala Gly Ala Thr Cys Cys Ala Cys Thr Gly Ala Thr GlyCys Gly 1585 1590 1595 1600 Thr Ala Thr Thr Cys Gly Ala Cys Gly Gly CysCys Gly Ala Ala Ala 1605 1610 1615 Thr Cys Gly Ala Gly Thr Ala Cys AlaAla Ala Thr Gly Gly Thr Gly 1620 1625 1630 Thr Ala Cys Gly Thr Cys GlyAla Ala Gly Gly Ala Gly Cys Cys Gly 1635 1640 1645 Ala Ala Thr Thr GlyThr Thr Cys Gly Ala Cys Ala Gly Cys Gly Gly 1650 1655 1660 Thr Cys AlaAla Gly Gly Cys Cys Gly Ala Cys Gly Cys Gly Ala Ala 1665 1670 1675 1680Cys Ala Thr Cys Gly Ala Ala Cys Thr Gly Thr Cys Gly Ala Gly Thr 16851690 1695 Thr Ala Thr Ala Ala Ala Thr Thr Cys Ala Cys Thr Ala Ala AlaAla 1700 1705 1710 Thr Cys Thr Gly Cys Cys Ala Ala Ala Ala Ala Cys GlyGly Ala Cys 1715 1720 1725 Ala Cys Thr Thr Gly Cys Cys Ala Gly Cys AlaCys Thr Thr Cys Ala 1730 1735 1740 Thr Cys Gly Gly Gly Gly Ala Cys CysThr Ala Cys Thr Cys Thr Cys 1745 1750 1755 1760 Gly Thr Cys Thr Ala CysGly Gly Gly Thr Thr Ala Gly Thr Thr Thr 1765 1770 1775 Cys Ala Thr AlaThr Thr Thr Gly Ala Thr Cys Gly Cys Gly Ala Cys 1780 1785 1790 Ala GlyCys Gly Gly Cys Thr Thr Cys Thr Ala Cys Thr Thr Thr Cys 1795 1800 1805Thr Thr Cys Ala Ala Ala Thr Ala Thr Thr Thr Thr Thr Cys Cys Cys 18101815 1820 Thr Gly Cys Cys Ala Gly Cys Cys Thr Cys Gly Thr Cys Gly ThrAla 1825 1830 1835 1840 Gly Thr Thr Thr Thr Ala Thr Cys Ala Thr Gly GlyAla Thr Cys Thr 1845 1850 1855 Cys Ala Thr Thr Cys Thr Gly Gly Ala ThrCys Ala Ala Thr Cys Gly 1860 1865 1870 Thr Gly Ala Cys Thr Cys Gly GlyCys Gly Cys Cys Thr Thr Cys Gly 1875 1880 1885 Cys Gly Ala Ala Cys CysCys Thr Ala Ala Thr Cys Gly Gly Thr Ala 1890 1895 1900 Cys Gly Ala ThrGly Ala Cys Gly Gly Thr Gly Cys Thr Cys Ala Cys 1905 1910 1915 1920 ThrGly Ala Gly Ala Cys Thr Cys Ala Thr Cys Thr Thr Ala Thr Gly 1925 19301935 Ala Cys Cys Gly Gly Ala Ala Cys Cys Ala Ala Thr Cys Gly Ala Cys1940 1945 1950 Gly Thr Cys Thr Thr Cys Cys Ala Cys Cys Ala Gly Thr ThrGly Cys 1955 1960 1965 Cys Thr Ala Thr Gly Thr Ala Ala Ala Ala Gly CysCys Gly Thr Thr 1970 1975 1980 Gly Ala Thr Gly Thr Ala Thr Thr Cys CysThr Cys Gly Gly Thr Thr 1985 1990 1995 2000 Thr Cys Thr Gly Cys Thr AlaThr Cys Thr Thr Cys Thr Gly Gly Thr 2005 2010 2015 Thr Ala Thr Ala CysThr Gly Gly Cys Gly Thr Thr Gly Ala Thr Cys 2020 2025 2030 Gly Ala GlyThr Ala Cys Gly Cys Cys Thr Gly Thr Gly Thr Thr Gly 2035 2040 2045 CysCys Thr Ala Cys Thr Cys Ala Ala Ala Ala Ala Ala Gly Ala Ala 2050 20552060 Gly Ala Ala Cys Gly Ala Gly Gly Ala Thr Cys Gly Thr Cys Gly Gly2065 2070 2075 2080 Ala Gly Ala Ala Gly Ala Gly Ala Gly Ala Ala Gly AlaAla Gly Ala 2085 2090 2095 Cys Gly Gly Ala Gly Cys Ala Thr Ala Ala AlaCys Cys Thr Gly Cys 2100 2105 2110 Thr Cys Cys Gly Cys Cys Gly Ala CysAla Cys Cys Thr Gly Ala Thr 2115 2120 2125 Ala Thr Thr Cys Thr Thr CysAla Cys Gly Ala Cys Gly Thr Cys Cys 2130 2135 2140 Gly Cys Cys Thr ThrGly Cys Cys Gly Ala Ala Thr Gly Cys Ala Cys 2145 2150 2155 2160 Ala ThrGly Cys Ala Ala Cys Gly Cys Gly Gly Cys Thr Cys Cys Ala 2165 2170 2175Ala Cys Cys Thr Cys Gly Ala Thr Cys Ala Thr Cys Gly Cys Cys Gly 21802185 2190 Thr Cys Ala Thr Cys Ala Ala Gly Cys Ala Gly Thr Cys Gly AlaAla 2195 2200 2205 Thr Cys Gly Ala Thr Thr Cys Thr Gly Thr Gly Thr CysAla Gly Thr 2210 2215 2220 Cys Ala Cys Ala Gly Thr Cys Ala Cys Ala ThrThr Gly Ala Cys Ala 2225 2230 2235 2240 Thr Cys Gly Thr Cys Ala Gly CysCys Gly Thr Gly Cys Cys Gly Cys 2245 2250 2255 Gly Thr Thr Thr Cys CysThr Cys Thr Thr Gly Thr Thr Thr Thr Cys 2260 2265 2270 Ala Thr Cys ThrThr Gly Thr Thr Cys Ala Ala Cys Ala Cys Thr Cys 2275 2280 2285 Thr CysThr Thr Cys Thr Gly Gly Cys Thr Gly Ala Thr Thr Cys Thr 2290 2295 2300Ala Cys Thr Gly Thr Ala Cys Ala Ala Ala Thr Cys Cys Ala Ala Gly 23052310 2315 2320 Cys Gly Thr Cys Thr Gly Cys Cys Gly Thr Ala Thr Ala ThrThr Ala 2325 2330 2335 Gly Thr Gly Ala Ala Cys Ala Cys Gly Ala Gly GlyGly Thr Gly Ala 2340 2345 2350 Cys Cys Gly Thr Thr Gly Cys Gly Ala ThrGly Cys Thr Cys Cys Ala 2355 2360 2365 Gly Ala Cys Cys Thr Thr Cys AlaThr Thr Ala Ala Thr Cys Thr Cys 2370 2375 2380 Ala Ala Thr Cys Cys AlaAla Cys Thr Thr Cys Cys Thr Cys Ala Thr 2385 2390 2395 2400 Cys Ala ThrThr Thr Thr Cys Cys Ala Thr Thr Thr Cys Gly Ala Ala 2405 2410 2415 ThrAla Thr Cys Thr Cys Thr Thr Thr Thr Thr Cys Thr Thr Gly Cys 2420 24252430 Ala Cys Ala Gly Ala Ala Gly Cys Cys Thr Thr Thr Thr Thr Thr Cys2435 2440 2445 Gly Thr Thr Thr Thr Thr Thr Thr Thr Thr Ala Thr Thr GlyAla Thr 2450 2455 2460 Thr Thr Ala Thr Thr Thr Thr Thr Ala Cys Gly GlyAla Thr Thr Thr 2465 2470 2475 2480 Thr Thr Ala Gly Ala Thr Ala Ala ThrGly Cys Ala Cys Ala Gly Ala 2485 2490 2495 Thr Gly Cys Cys Thr Cys AlaThr Thr Gly Cys Thr Cys Ala Ala Ala 2500 2505 2510 Thr Ala Ala Ala ThrThr Thr Ala Thr Thr Thr Thr Ala Ala Thr Thr 2515 2520 2525 Ala Ala AlaAla Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 2530 2535 2540 42544 DNA Caenorhabditis elegans 4 aagtttgaga gtgatatagg agaaaaacctccccaacatt ggctcacacc cggattatga 60 tcttctgctg ctcctgctgc tccttctgctgtagttgaga cgaagaagaa gaagaagctc 120 cattctcgag aaatggctcg tccattcacacttatcgtac tcctctccgc acatctgtgt 180 ctacatgtgg ttgtgacaca ggatgaggactcacatatca acactcaact cctctcatca 240 gttctcgata gactcacgaa tcgcactacttatgataaaa gattacggcc caggtatggt 300 gaaaagccag tcgacgttgg aattacgatacacgtttctt caatctctgc agtttcagaa 360 gttgatatgg acttcacatt agacttctacatgcgtcaaa cgtggcaaga ccctcgacta 420 gccttcggaa gtcttgattt gggactttccaaagaaatcg actcacttac cgtcggagta 480 gactacctgg atagactgtg gaaacccgacacgttcttcc caaatgaaaa gaaatcattc 540 ttccacttgg caaccacaca taactcgttccttcgtatcg agggtgatgg aacggtttat 600 actagtcaaa gattaacagt cactgcaacgtgtccaatgg acctgaagct gttcccaatg 660 gactctcaac actgtaaact ggaaattgaaagctacgggt acgagacgaa agatatcgac 720 tactattggg ggaagaagcg gactgatttggagataacgg ctgtcaagtt tgataccttc 780 cagttgccgc agtttcagcc aacgctgtattttgtgaata caactaaagc cgagacctca 840 tcaggaaaat acgtacgcct ggcgctggaagtaatattgg ttcgaaatat gggcttctac 900 actatgaaca tcgtcatccc atccatcctgatcgtcacca tatcttgggt atcattttgg 960 ttgaatcgag aagcttcgcc ggctcgagttggattgggtg tgactactgt gctcacaatg 1020 acaactctga tcactacaac caataattcgatgccaaaag tgtcttatgt caagggtctg 1080 gatgtgtttc ttaatttttg tttcgtaatggtattcgcct cgttgctcga gtacgccata 1140 gtatcctaca tgaataaacg actggtcctccgacgggaaa aacgaagaaa agccgccgaa 1200 caacagcagc gaaacgagat gccaatgttcaacgcgagcc cgaaggccgc caataataat 1260 gctgacttgt actttgccgg acacaattcctctatgaatc cattgatgga gatcccagaa 1320 aattgtgatt gccggacgat tccaatgatgcaacatccac gtcttgtcac agacggcgca 1380 catacgctat ggccggctcc attcgcgcggccgaaaaagg cttccaagac atgctgccaa 1440 cgatggacgc ctgcaaaaat cgataagcttagccgatacg gtttcccatt gtctttctct 1500 atcttcaata tagtctactg gttgtatatgaaatatctaa gcttaaactc gtcggacaag 1560 atccaggaga acgacaagtg gcagcagatccactgatgcg tattcgacgg ccgaaatcga 1620 gtacaaatgg tgtacgtcga aggagccgaattgttcgaca gcggtcaagg ccgacgcgaa 1680 catcgaactg tcgagttata aattcactaaaatctgccaa aaacggacac ttgccagcac 1740 ttcatcgggg acctactctc gtctacgggttagtttcata tttgatcgcg acagcggctt 1800 ctactttctt caaatatttt tccctgccagcctcgtcgta gttttatcat ggatctcatt 1860 ctggatcaat cgtgactcgg cgccttcgcgaaccctaatc ggtacgatga cggtgctcac 1920 tgagactcat cttatgaccg gaaccaatcgacgtcttcca ccagttgcct atgtaaaagc 1980 cgttgatgta ttcctcggtt tctgctatcttctggttata ctggcgttga tcgagtacgc 2040 ctgtgttgcc tactcaaaaa agaagaacgaggatcgtcgg agaagagaga agaagacgga 2100 gcataaacct gctccgccga cacctgatattcttcacgac gtccgccttg ccgaatgcac 2160 atgcaacgcg gctccaacct cgatcatcgccgtcatcaag cagtcgaatc gattctgtgt 2220 cagtcacagt cacattgaca tcgtcagccgtgccgcgttt cctcttgttt tcatcttgtt 2280 caacactctc ttctggctga ttctactgtacaaatccaag cgtctgccgt atattagtga 2340 acacgagggt gaccgttgcg atgctccagaccttcattaa tctcaatcca acttcctcat 2400 cattttccat ttcgaatatc tctttttcttgcacagaagc cttttttcgt ttttttttat 2460 tgatttattt ttacggattt ttagataatgcacagatgcc tcattgctca aataaattta 2520 ttttaattaa aaaaaaaaaa aaaa 2544 51917 PRT Caenorhabditis elegans 5 Gly Ala Thr Cys Cys Cys Cys Ala GlyCys Gly Cys Cys Thr Cys Cys 1 5 10 15 Cys Cys Gly Thr Thr Ala Cys CysThr Cys Thr Gly Thr Gly Thr Thr 20 25 30 Cys Thr Cys Cys Gly Thr Gly ThrThr Gly Gly Cys Thr Cys Ala Ala 35 40 45 Cys Thr Thr Gly Thr Gly Cys GlyThr Gly Thr Thr Thr Thr Ala Cys 50 55 60 Ala Thr Cys Thr Cys Thr Cys ThrGly Thr Cys Thr Cys Thr Cys Thr 65 70 75 80 Cys Thr Cys Ala Thr Cys AlaThr Cys Ala Thr Cys Ala Thr Cys Thr 85 90 95 Thr Ala Thr Thr Cys Thr GlyThr Gly Cys Thr Cys Cys Cys Gly Cys 100 105 110 Thr Ala Ala Cys Cys GlyAla Thr Thr Gly Gly Cys Thr Cys Ala Thr 115 120 125 Thr Cys Thr Cys ThrThr Cys Gly Gly Cys Thr Cys Thr Thr Gly Thr 130 135 140 Cys Ala Cys ThrAla Ala Cys Gly Ala Ala Thr Cys Gly Cys Thr Thr 145 150 155 160 Thr ThrCys Ala Cys Ala Cys Ala Gly Ala Gly Thr Gly Ala Thr Ala 165 170 175 ThrAla Gly Gly Ala Gly Ala Ala Ala Ala Ala Cys Cys Thr Cys Cys 180 185 190Cys Cys Ala Ala Cys Ala Thr Thr Gly Gly Cys Thr Cys Ala Cys Ala 195 200205 Cys Cys Cys Gly Gly Ala Thr Thr Ala Thr Gly Ala Thr Cys Thr Thr 210215 220 Cys Thr Gly Cys Thr Gly Cys Thr Cys Cys Thr Gly Cys Thr Gly Cys225 230 235 240 Thr Cys Cys Thr Thr Cys Thr Gly Cys Thr Gly Thr Ala GlyThr Thr 245 250 255 Gly Ala Gly Ala Cys Gly Ala Ala Gly Ala Ala Gly AlaAla Gly Ala 260 265 270 Ala Gly Ala Ala Gly Cys Thr Cys Cys Ala Thr ThrCys Thr Cys Gly 275 280 285 Ala Gly Ala Ala Ala Thr Gly Gly Cys Thr CysGly Thr Cys Cys Ala 290 295 300 Thr Thr Cys Ala Cys Ala Cys Thr Thr AlaThr Cys Gly Thr Ala Cys 305 310 315 320 Thr Cys Cys Thr Cys Thr Cys CysGly Cys Ala Cys Ala Thr Cys Thr 325 330 335 Gly Thr Gly Thr Cys Thr AlaCys Ala Thr Gly Thr Gly Gly Thr Thr 340 345 350 Gly Thr Gly Ala Cys AlaCys Ala Gly Gly Ala Thr Gly Ala Gly Gly 355 360 365 Ala Cys Thr Cys AlaCys Ala Thr Ala Thr Cys Ala Ala Cys Ala Cys 370 375 380 Thr Cys Ala AlaCys Thr Cys Cys Thr Cys Thr Cys Ala Thr Cys Ala 385 390 395 400 Gly ThrThr Cys Thr Cys Gly Ala Thr Ala Gly Ala Cys Thr Cys Ala 405 410 415 CysGly Ala Ala Thr Cys Gly Cys Ala Cys Thr Ala Cys Thr Thr Ala 420 425 430Thr Gly Ala Thr Ala Ala Ala Ala Gly Ala Thr Thr Ala Cys Gly Gly 435 440445 Cys Cys Cys Ala Gly Gly Thr Ala Thr Gly Gly Thr Gly Ala Ala Ala 450455 460 Ala Gly Cys Cys Ala Gly Thr Cys Gly Ala Cys Gly Thr Thr Gly Gly465 470 475 480 Ala Ala Thr Thr Ala Cys Gly Ala Thr Ala Cys Ala Cys GlyThr Thr 485 490 495 Thr Cys Thr Thr Cys Ala Ala Thr Cys Thr Cys Thr GlyCys Ala Gly 500 505 510 Thr Thr Thr Cys Ala Gly Ala Ala Gly Thr Thr GlyAla Thr Ala Thr 515 520 525 Gly Gly Ala Cys Thr Thr Cys Ala Cys Ala ThrThr Ala Gly Ala Cys 530 535 540 Thr Thr Cys Thr Ala Cys Ala Thr Gly CysGly Thr Cys Ala Ala Ala 545 550 555 560 Cys Gly Thr Gly Gly Cys Ala AlaGly Ala Cys Cys Cys Thr Cys Gly 565 570 575 Ala Cys Thr Ala Gly Cys CysThr Thr Cys Gly Gly Ala Ala Gly Thr 580 585 590 Cys Thr Thr Gly Ala ThrThr Thr Gly Gly Gly Ala Cys Thr Thr Thr 595 600 605 Cys Cys Ala Ala AlaGly Ala Ala Ala Thr Cys Gly Ala Cys Thr Cys 610 615 620 Ala Cys Thr ThrAla Cys Cys Gly Thr Cys Gly Gly Ala Gly Thr Ala 625 630 635 640 Gly AlaCys Thr Ala Cys Cys Thr Gly Gly Ala Thr Ala Gly Ala Cys 645 650 655 ThrGly Thr Gly Gly Ala Ala Ala Cys Cys Cys Gly Ala Cys Ala Cys 660 665 670Gly Thr Thr Cys Thr Thr Cys Cys Cys Ala Ala Ala Thr Gly Ala Ala 675 680685 Ala Ala Gly Ala Ala Ala Thr Cys Ala Thr Thr Cys Thr Thr Cys Cys 690695 700 Ala Cys Thr Thr Gly Gly Cys Ala Ala Cys Cys Ala Cys Ala Cys Ala705 710 715 720 Thr Ala Ala Cys Thr Cys Gly Thr Thr Cys Cys Thr Thr CysGly Thr 725 730 735 Ala Thr Cys Gly Ala Gly Gly Gly Thr Gly Ala Thr GlyGly Ala Ala 740 745 750 Cys Gly Gly Thr Thr Thr Ala Thr Ala Cys Thr AlaGly Thr Cys Ala 755 760 765 Ala Ala Gly Ala Thr Thr Ala Ala Cys Ala GlyThr Cys Ala Cys Thr 770 775 780 Gly Cys Ala Ala Cys Gly Thr Gly Thr CysCys Ala Ala Thr Gly Gly 785 790 795 800 Ala Cys Cys Thr Gly Ala Ala GlyCys Thr Gly Thr Thr Cys Cys Cys 805 810 815 Ala Ala Thr Gly Gly Ala CysThr Cys Thr Cys Ala Ala Cys Ala Cys 820 825 830 Thr Gly Thr Ala Ala AlaCys Thr Gly Gly Ala Ala Ala Thr Thr Gly 835 840 845 Ala Ala Ala Gly CysThr Ala Cys Gly Gly Cys Thr Ala Cys Ala Gly 850 855 860 Thr Ala Thr CysCys Thr Cys Gly Ala Cys Ala Thr Thr Ala Thr Gly 865 870 875 880 Thr AlaCys Gly Thr Gly Thr Cys Gly Cys Ala Cys Gly Ala Gly Ala 885 890 895 AlaGly Ala Ala Gly Thr Cys Cys Gly Thr Gly Thr Cys Cys Ala Cys 900 905 910Cys Gly Ala Gly Thr Cys Thr Thr Ala Thr Gly Ala Gly Thr Thr Gly 915 920925 Cys Cys Gly Cys Ala Gly Thr Thr Thr Gly Thr Ala Cys Thr Thr Cys 930935 940 Ala Gly Thr Cys Thr Ala Thr Cys Ala Ala Gly Gly Thr Cys Gly Thr945 950 955 960 Cys Ala Ala Thr Cys Ala Thr Ala Cys Gly Cys Ala Ala AlaAla Gly 965 970 975 Cys Thr Thr Ala Gly Thr Thr Cys Ala Gly Gly Ala GlyAla Ala Thr 980 985 990 Ala Thr Thr Cys Cys Cys Gly Cys Cys Thr Thr ThrGly Cys Thr Gly 995 1000 1005 Gly Thr Thr Cys Thr Thr Cys Cys Thr AlaThr Thr Cys Ala Ala Gly 1010 1015 1020 Cys Gly Thr Ala Ala Cys Ala ThrCys Gly Gly Cys Thr Thr Cys Thr 1025 1030 1035 1040 Ala Cys Ala Thr CysAla Thr Cys Cys Ala Ala Ala Thr Ala Thr Ala 1045 1050 1055 Thr Cys ThrAla Cys Cys Ala Thr Cys Thr Gly Thr Cys Cys Thr Gly 1060 1065 1070 AlaThr Thr Gly Thr Cys Gly Thr Cys Ala Thr Cys Thr Cys Ala Thr 1075 10801085 Gly Gly Gly Thr Ala Thr Cys Thr Thr Thr Thr Thr Gly Gly Thr Thr1090 1095 1100 Gly Ala Gly Cys Cys Gly Cys Gly Ala Thr Gly Cys Gly AlaCys Ala 1105 1110 1115 1120 Cys Cys Gly Gly Cys Ala Ala Gly Ala Gly ThrThr Gly Cys Thr Cys 1125 1130 1135 Thr Cys Gly Gly Ala Gly Thr Cys AlaCys Cys Ala Cys Thr Gly Thr 1140 1145 1150 Gly Cys Thr Cys Ala Cys AlaAla Thr Gly Ala Cys Thr Ala Cys Thr 1155 1160 1165 Thr Thr Gly Ala ThrGly Ala Cys Cys Ala Thr Gly Ala Cys Thr Ala 1170 1175 1180 Ala Thr AlaGly Thr Thr Cys Ala Ala Thr Gly Cys Cys Ala Ala Ala 1185 1190 1195 1200Ala Gly Thr Gly Thr Cys Ala Thr Ala Thr Gly Thr Gly Ala Ala Ala 12051210 1215 Ala Gly Thr Ala Thr Cys Gly Ala Thr Ala Thr Ala Thr Thr ThrCys 1220 1225 1230 Thr Ala Gly Gly Thr Gly Thr Cys Thr Gly Cys Thr ThrCys Ala Thr 1235 1240 1245 Gly Ala Thr Gly Gly Thr Ala Thr Thr Cys ThrGly Thr Thr Cys Ala 1250 1255 1260 Cys Thr Thr Cys Thr Ala Gly Ala AlaThr Ala Cys Gly Cys Cys Gly 1265 1270 1275 1280 Cys Cys Gly Thr Cys GlyGly Ala Thr Ala Cys Ala Thr Cys Ala Gly 1285 1290 1295 Cys Ala Ala AlaCys Gly Gly Ala Thr Gly Ala Ala Gly Cys Thr Thr 1300 1305 1310 Gly ThrCys Cys Gly Ala Gly Cys Cys Ala Gly Ala Ala Ala Ala Gly 1315 1320 1325Ala Ala Thr Cys Thr Cys Gly Ala Ala Thr Gly Cys Thr Gly Ala Cys 13301335 1340 Cys Cys Cys Thr Thr Thr Ala Cys Cys Ala Cys Ala Thr Cys ThrThr 1345 1350 1355 1360 Gly Ala Gly Thr Cys Thr Cys Thr Thr Cys Cys ThrCys Cys Ala Ala 1365 1370 1375 Ala Ala Cys Gly Thr Ala Cys Thr Cys ThrAla Thr Cys Cys Gly Thr 1380 1385 1390 Thr Cys Cys Cys Thr Cys Gly ThrAla Thr Thr Thr Cys Ala Ala Cys 1395 1400 1405 Ala Ala Cys Ala Cys CysAla Cys Gly Thr Ala Cys Cys Gly Cys Cys 1410 1415 1420 Cys Gly Thr ThrThr Thr Ala Cys Thr Cys Gly Thr Cys Cys Ala Cys 1425 1430 1435 1440 CysGly Ala Thr Cys Ala Ala Ala Cys Gly Thr Cys Cys Ala Ala Cys 1445 14501455 Cys Thr Gly Thr Ala Cys Ala Thr Thr Cys Cys Gly Gly Ala Gly Thr1460 1465 1470 Cys Gly Cys Ala Gly Cys Gly Cys Ala Cys Gly Ala Cys GlyAla Thr 1475 1480 1485 Thr Thr Thr Cys Thr Cys Ala Ala Ala Thr Gly AlaGly Gly Ala Thr 1490 1495 1500 Gly Cys Ala Gly Thr Gly Cys Cys Gly AlaAla Thr Gly Ala Ala Cys 1505 1510 1515 1520 Thr Ala Ala Cys Thr Cys CysAla Ala Thr Gly Cys Thr Cys Gly Gly 1525 1530 1535 Ala Cys Gly Gly AlaGly Thr Ala Ala Cys Thr Cys Ala Cys Ala Ala 1540 1545 1550 Gly Cys AlaThr Cys Cys Gly Thr Ala Thr Thr Thr Cys Thr Gly Thr 1555 1560 1565 AlaThr Cys Ala Gly Ala Cys Gly Gly Cys Thr Gly Thr Ala Ala Thr 1570 15751580 Ala Thr Cys Cys Gly Ala Thr Gly Ala Cys Gly Ala Gly Thr Thr Cys1585 1590 1595 1600 Gly Gly Ala Ala Gly Ala Thr Thr Cys Thr Gly Gly CysGly Thr Thr 1605 1610 1615 Gly Gly Cys Thr Cys Cys Gly Ala Cys Cys AlaThr Cys Cys Ala Ala 1620 1625 1630 Cys Ala Thr Thr Gly Ala Cys Ala AlaGly Thr Ala Cys Thr Cys Ala 1635 1640 1645 Cys Gly Cys Thr Cys Ala CysThr Gly Thr Thr Cys Cys Cys Ala Thr 1650 1655 1660 Cys Thr Ala Thr ThrThr Thr Thr Gly Thr Gly Cys Thr Cys Thr Thr 1665 1670 1675 1680 Cys AlaAla Cys Gly Thr Cys Gly Gly Cys Thr Ala Cys Thr Gly Gly 1685 1690 1695Gly Cys Cys Thr Ala Cys Thr Thr Cys Ala Thr Cys Cys Gly Gly Cys 17001705 1710 Ala Gly Ala Gly Cys Cys Ala Gly Ala Thr Thr Cys Ala Gly GlyAla 1715 1720 1725 Ala Gly Ala Gly Cys Ala Ala Cys Gly Gly Ala Ala CysAla Gly Thr 1730 1735 1740 Cys Ala Ala Ala Thr Thr Cys Thr Cys Thr AlaAla Thr Thr Thr Cys 1745 1750 1755 1760 Thr Gly Ala Thr Cys Ala Cys AlaCys Cys Ala Cys Thr Cys Cys Thr 1765 1770 1775 Cys Ala Thr Cys Thr CysAla Thr Thr Cys Thr Ala Thr Thr Gly Thr 1780 1785 1790 Ala Gly Cys CysThr Thr Thr Thr Thr Thr Thr Thr Thr Cys Gly Ala 1795 1800 1805 Ala ThrCys Ala Thr Thr Thr Cys Thr Gly Ala Ala Thr Ala Thr Cys 1810 1815 1820Thr Cys Thr Thr Ala Thr Cys Thr Thr Cys Thr Cys Ala Ala Ala Gly 18251830 1835 1840 Ala Thr Gly Gly Cys Ala Thr Cys Gly Cys Cys Thr Ala AlaCys Cys 1845 1850 1855 Ala Cys Cys Cys Cys Cys Cys Ala Cys Thr Thr AlaGly Cys Ala Cys 1860 1865 1870 Ala Cys Ala Ala Ala Ala Ala Thr Gly CysThr Cys Thr Cys Thr Thr 1875 1880 1885 Cys Thr Ala Gly Ala Ala Thr ThrThr Gly Thr Thr Gly Ala Gly Ala 1890 1895 1900 Ala Cys Gly Gly Ala GlyAla Cys Ala Cys Cys Gly Ala 1905 1910 1915 6 1917 DNA Caenorhabditiselegans 6 gatccccagc gcctccccgt tacctctgtg ttctccgtgt tggctcaacttgtgcgtgtt 60 ttacatctct ctgtctctct ctcatcatca tcatcttatt ctgtgctcccgctaaccgat 120 tggctcattc tcttcggctc ttgtcactaa cgaatcgctt ttcacacagagtgatatagg 180 agaaaaacct ccccaacatt ggctcacacc cggattatga tcttctgctgctcctgctgc 240 tccttctgct gtagttgaga cgaagaagaa gaagaagctc cattctcgagaaatggctcg 300 tccattcaca cttatcgtac tcctctccgc acatctgtgt ctacatgtggttgtgacaca 360 ggatgaggac tcacatatca acactcaact cctctcatca gttctcgatagactcacgaa 420 tcgcactact tatgataaaa gattacggcc caggtatggt gaaaagccagtcgacgttgg 480 aattacgata cacgtttctt caatctctgc agtttcagaa gttgatatggacttcacatt 540 agacttctac atgcgtcaaa cgtggcaaga ccctcgacta gccttcggaagtcttgattt 600 gggactttcc aaagaaatcg actcacttac cgtcggagta gactacctggatagactgtg 660 gaaacccgac acgttcttcc caaatgaaaa gaaatcattc ttccacttggcaaccacaca 720 taactcgttc cttcgtatcg agggtgatgg aacggtttat actagtcaaagattaacagt 780 cactgcaacg tgtccaatgg acctgaagct gttcccaatg gactctcaacactgtaaact 840 ggaaattgaa agctacggct acagtatcct cgacattatg tacgtgtcgcacgagaagaa 900 gtccgtgtcc accgagtctt atgagttgcc gcagtttgta cttcagtctatcaaggtcgt 960 caatcatacg caaaagctta gttcaggaga atattcccgc ctttgctggttcttcctatt 1020 caagcgtaac atcggcttct acatcatcca aatatatcta ccatctgtcctgattgtcgt 1080 catctcatgg gtatcttttt ggttgagccg cgatgcgaca ccggcaagagttgctctcgg 1140 agtcaccact gtgctcacaa tgactacttt gatgaccatg actaatagttcaatgccaaa 1200 agtgtcatat gtgaaaagta tcgatatatt tctaggtgtc tgcttcatgatggtattctg 1260 ttcacttcta gaatacgccg ccgtcggata catcagcaaa cggatgaagcttgtccgagc 1320 cagaaaagaa tctcgaatgc tgaccccttt accacatctt gagtctcttcctccaaaacg 1380 tactctatcc gttccctcgt atttcaacaa caccacgtac cgcccgttttactcgtccac 1440 cgatcaaacg tccaacctgt acattccgga gtcgcagcgc acgacgattttctcaaatga 1500 ggatgcagtg ccgaatgaac taactccaat gctcggacgg agtaactcacaagcatccgt 1560 atttctgtat cagacggctg taatatccga tgacgagttc ggaagattctggcgttggct 1620 ccgaccatcc aacattgaca agtactcacg ctcactgttc ccatctatttttgtgctctt 1680 caacgtcggc tactgggcct acttcatccg gcagagccag attcaggaagagcaacggaa 1740 cagtcaaatt ctctaatttc tgatcacacc actcctcatc tcattctattgtagcctttt 1800 tttttcgaat catttctgaa tatctcttat cttctcaaag atggcatcgcctaaccaccc 1860 cccacttagc acacaaaaat gctctcttct agaatttgtt gagaacggagacaccga 1917 7 2508 PRT Caenorhabditis elegans 7 Ala Ala Gly Thr Thr ThrGly Ala Gly Ala Gly Thr Gly Ala Thr Ala 1 5 10 15 Thr Ala Gly Gly AlaGly Ala Ala Ala Ala Ala Cys Cys Thr Cys Cys 20 25 30 Cys Cys Ala Ala CysAla Thr Thr Gly Gly Cys Thr Cys Ala Cys Ala 35 40 45 Cys Cys Cys Gly GlyAla Thr Thr Ala Thr Gly Ala Thr Cys Thr Thr 50 55 60 Cys Thr Gly Cys ThrGly Cys Thr Cys Cys Thr Gly Cys Thr Gly Cys 65 70 75 80 Thr Cys Cys ThrThr Cys Thr Gly Cys Thr Gly Thr Ala Gly Thr Thr 85 90 95 Gly Ala Gly AlaCys Gly Ala Ala Gly Ala Ala Gly Ala Ala Gly Ala 100 105 110 Ala Gly AlaAla Gly Cys Thr Cys Cys Ala Thr Thr Cys Thr Cys Gly 115 120 125 Ala GlyAla Ala Ala Thr Gly Gly Cys Thr Cys Gly Thr Cys Cys Ala 130 135 140 ThrThr Cys Ala Cys Ala Cys Thr Thr Ala Thr Cys Gly Thr Ala Cys 145 150 155160 Thr Cys Cys Thr Cys Thr Cys Cys Gly Cys Ala Cys Ala Thr Cys Thr 165170 175 Gly Thr Gly Thr Cys Thr Ala Cys Ala Thr Gly Thr Gly Gly Thr Thr180 185 190 Gly Thr Gly Ala Cys Ala Cys Ala Gly Gly Ala Thr Gly Ala GlyGly 195 200 205 Ala Cys Thr Cys Ala Cys Ala Thr Ala Thr Cys Ala Ala CysAla Cys 210 215 220 Thr Cys Ala Ala Cys Thr Cys Cys Thr Cys Thr Cys AlaThr Cys Ala 225 230 235 240 Gly Thr Thr Cys Thr Cys Gly Ala Thr Ala GlyAla Cys Thr Cys Ala 245 250 255 Cys Gly Ala Ala Thr Cys Gly Cys Ala CysThr Ala Cys Thr Thr Ala 260 265 270 Thr Gly Ala Thr Ala Ala Ala Ala GlyAla Thr Thr Ala Cys Gly Gly 275 280 285 Cys Cys Cys Ala Gly Gly Thr AlaThr Gly Gly Thr Gly Ala Ala Ala 290 295 300 Ala Gly Cys Cys Ala Gly ThrCys Gly Ala Cys Gly Thr Thr Gly Gly 305 310 315 320 Ala Ala Thr Thr AlaCys Gly Ala Thr Ala Cys Ala Cys Gly Thr Thr 325 330 335 Thr Cys Thr ThrCys Ala Ala Thr Cys Thr Cys Thr Gly Cys Ala Gly 340 345 350 Thr Thr ThrCys Ala Gly Ala Ala Gly Thr Thr Gly Ala Thr Ala Thr 355 360 365 Gly GlyAla Cys Thr Thr Cys Ala Cys Ala Thr Thr Ala Gly Ala Cys 370 375 380 ThrThr Cys Thr Ala Cys Ala Thr Gly Cys Gly Thr Cys Ala Ala Ala 385 390 395400 Cys Gly Thr Gly Gly Cys Ala Ala Gly Ala Cys Cys Cys Thr Cys Gly 405410 415 Ala Cys Thr Ala Gly Cys Cys Thr Thr Cys Gly Gly Ala Ala Gly Thr420 425 430 Cys Thr Thr Gly Ala Thr Thr Thr Gly Gly Gly Ala Cys Thr ThrThr 435 440 445 Cys Cys Ala Ala Ala Gly Ala Ala Ala Thr Cys Gly Ala CysThr Cys 450 455 460 Ala Cys Thr Thr Ala Cys Cys Gly Thr Cys Gly Gly AlaGly Thr Ala 465 470 475 480 Gly Ala Cys Thr Ala Cys Cys Thr Gly Gly AlaThr Ala Gly Ala Cys 485 490 495 Thr Gly Thr Gly Gly Ala Ala Ala Cys CysCys Gly Ala Cys Ala Cys 500 505 510 Gly Thr Thr Cys Thr Thr Cys Cys CysAla Ala Ala Thr Gly Ala Ala 515 520 525 Ala Ala Gly Ala Ala Ala Thr CysAla Thr Thr Cys Thr Thr Cys Cys 530 535 540 Ala Cys Thr Thr Gly Gly CysAla Ala Cys Cys Ala Cys Ala Cys Ala 545 550 555 560 Thr Ala Ala Cys ThrCys Gly Thr Thr Cys Cys Thr Thr Cys Gly Thr 565 570 575 Ala Thr Cys GlyAla Gly Gly Gly Thr Gly Ala Thr Gly Gly Ala Ala 580 585 590 Cys Gly GlyThr Thr Thr Ala Thr Ala Cys Thr Ala Gly Thr Cys Ala 595 600 605 Ala AlaGly Ala Thr Thr Ala Ala Cys Ala Gly Thr Cys Ala Cys Thr 610 615 620 GlyCys Ala Ala Cys Gly Thr Gly Thr Cys Cys Ala Ala Thr Gly Gly 625 630 635640 Ala Cys Cys Thr Gly Ala Ala Gly Cys Thr Gly Thr Thr Cys Cys Cys 645650 655 Ala Ala Thr Gly Gly Ala Cys Thr Cys Thr Cys Ala Ala Cys Ala Cys660 665 670 Thr Gly Thr Ala Ala Ala Cys Thr Gly Gly Ala Ala Ala Thr ThrGly 675 680 685 Ala Ala Ala Gly Cys Thr Ala Cys Gly Gly Gly Thr Ala CysGly Ala 690 695 700 Gly Ala Cys Gly Ala Ala Ala Gly Ala Thr Ala Thr CysGly Ala Cys 705 710 715 720 Thr Ala Cys Thr Ala Thr Thr Gly Gly Gly GlyGly Ala Ala Gly Ala 725 730 735 Ala Gly Cys Gly Gly Ala Cys Thr Gly AlaThr Thr Thr Gly Gly Ala 740 745 750 Gly Ala Thr Ala Ala Cys Gly Gly CysThr Gly Thr Cys Ala Ala Gly 755 760 765 Thr Thr Thr Gly Ala Thr Ala CysCys Thr Thr Cys Cys Ala Gly Thr 770 775 780 Thr Gly Cys Cys Gly Cys AlaGly Thr Thr Thr Cys Ala Gly Cys Cys 785 790 795 800 Ala Ala Cys Gly CysThr Gly Thr Ala Thr Thr Thr Thr Gly Thr Gly 805 810 815 Ala Ala Thr AlaCys Ala Ala Cys Thr Ala Ala Ala Gly Cys Cys Gly 820 825 830 Ala Gly AlaCys Cys Thr Cys Ala Thr Cys Ala Gly Gly Ala Ala Ala 835 840 845 Ala ThrAla Cys Gly Thr Ala Cys Gly Cys Cys Thr Gly Gly Cys Gly 850 855 860 CysThr Gly Gly Ala Ala Gly Thr Ala Ala Thr Ala Thr Thr Gly Gly 865 870 875880 Thr Thr Cys Gly Ala Ala Ala Thr Ala Thr Gly Gly Gly Cys Thr Thr 885890 895 Cys Thr Ala Cys Ala Cys Thr Ala Thr Gly Ala Ala Cys Ala Thr Cys900 905 910 Gly Thr Cys Ala Thr Cys Cys Cys Ala Thr Cys Cys Ala Thr CysCys 915 920 925 Thr Gly Ala Thr Cys Gly Thr Cys Ala Cys Cys Ala Thr AlaThr Cys 930 935 940 Thr Thr Gly Gly Gly Thr Ala Thr Cys Ala Thr Thr ThrThr Gly Gly 945 950 955 960 Thr Thr Gly Ala Ala Thr Cys Gly Ala Gly AlaAla Gly Cys Thr Thr 965 970 975 Cys Gly Cys Cys Gly Gly Cys Thr Cys GlyAla Gly Thr Thr Gly Gly 980 985 990 Ala Thr Thr Gly Gly Gly Thr Gly ThrGly Ala Cys Thr Ala Cys Thr 995 1000 1005 Gly Thr Gly Cys Thr Cys AlaCys Ala Ala Thr Gly Ala Cys Ala Ala 1010 1015 1020 Cys Thr Cys Thr GlyAla Thr Cys Ala Cys Thr Ala Cys Ala Ala Cys 1025 1030 1035 1040 Cys AlaAla Thr Ala Ala Thr Thr Cys Gly Ala Thr Gly Cys Cys Ala 1045 1050 1055Ala Ala Ala Gly Thr Gly Thr Cys Thr Thr Ala Thr Gly Thr Cys Ala 10601065 1070 Ala Gly Gly Gly Thr Cys Thr Gly Gly Ala Thr Gly Thr Gly ThrThr 1075 1080 1085 Thr Cys Thr Thr Ala Ala Thr Thr Thr Thr Thr Gly ThrThr Thr Cys 1090 1095 1100 Gly Thr Ala Ala Thr Gly Gly Thr Ala Thr ThrCys Gly Cys Cys Thr 1105 1110 1115 1120 Cys Gly Thr Thr Gly Cys Thr CysGly Ala Gly Thr Ala Cys Gly Cys 1125 1130 1135 Cys Ala Thr Ala Gly ThrAla Thr Cys Cys Thr Ala Cys Ala Thr Gly 1140 1145 1150 Ala Ala Thr AlaAla Ala Cys Gly Ala Cys Thr Gly Gly Thr Cys Cys 1155 1160 1165 Thr CysCys Gly Ala Cys Gly Gly Gly Ala Ala Ala Ala Ala Cys Gly 1170 1175 1180Ala Ala Gly Ala Ala Ala Ala Gly Cys Cys Gly Cys Cys Gly Ala Ala 11851190 1195 1200 Cys Ala Ala Cys Ala Gly Cys Ala Gly Cys Gly Ala Ala AlaCys Gly 1205 1210 1215 Ala Gly Ala Thr Gly Cys Cys Ala Ala Thr Gly ThrThr Cys Ala Ala 1220 1225 1230 Cys Gly Cys Gly Ala Gly Cys Cys Cys GlyAla Ala Gly Gly Cys Cys 1235 1240 1245 Gly Cys Cys Ala Ala Thr Ala AlaThr Ala Ala Thr Ala Ala Thr Cys 1250 1255 1260 Cys Ala Thr Thr Gly AlaThr Gly Gly Ala Gly Ala Thr Cys Cys Cys 1265 1270 1275 1280 Ala Gly AlaAla Ala Ala Thr Thr Gly Thr Gly Ala Thr Thr Gly Cys 1285 1290 1295 CysGly Gly Ala Cys Gly Ala Thr Thr Cys Cys Ala Ala Thr Gly Ala 1300 13051310 Thr Gly Cys Ala Ala Cys Ala Thr Cys Cys Ala Cys Gly Thr Cys Thr1315 1320 1325 Thr Gly Thr Cys Ala Cys Ala Gly Ala Cys Gly Gly Cys GlyCys Ala 1330 1335 1340 Cys Ala Thr Ala Cys Gly Cys Thr Ala Thr Gly GlyCys Cys Gly Gly 1345 1350 1355 1360 Cys Thr Cys Cys Ala Thr Thr Cys GlyCys Gly Cys Gly Gly Cys Cys 1365 1370 1375 Gly Ala Ala Ala Ala Ala GlyGly Cys Thr Thr Cys Cys Ala Ala Gly 1380 1385 1390 Ala Cys Ala Thr GlyCys Thr Gly Cys Cys Ala Ala Cys Gly Ala Thr 1395 1400 1405 Gly Gly AlaCys Gly Cys Cys Thr Gly Cys Ala Ala Ala Ala Ala Thr 1410 1415 1420 CysGly Ala Thr Ala Ala Gly Cys Thr Thr Ala Gly Cys Cys Gly Ala 1425 14301435 1440 Thr Ala Cys Gly Gly Thr Thr Thr Cys Cys Cys Ala Thr Thr GlyThr 1445 1450 1455 Cys Thr Thr Thr Cys Thr Cys Thr Ala Thr Cys Thr ThrCys Ala Ala 1460 1465 1470 Thr Ala Thr Ala Gly Thr Cys Thr Ala Cys ThrGly Gly Thr Thr Gly 1475 1480 1485 Thr Ala Thr Ala Thr Gly Ala Ala AlaThr Ala Thr Cys Thr Ala Ala 1490 1495 1500 Gly Cys Thr Thr Ala Ala AlaCys Thr Cys Gly Thr Cys Gly Gly Ala 1505 1510 1515 1520 Cys Ala Ala GlyAla Thr Cys Cys Ala Gly Gly Ala Gly Ala Ala Cys 1525 1530 1535 Gly AlaCys Ala Ala Gly Thr Gly Gly Cys Ala Gly Cys Ala Gly Ala 1540 1545 1550Thr Cys Cys Ala Cys Thr Gly Ala Thr Gly Cys Gly Thr Ala Thr Thr 15551560 1565 Cys Gly Ala Cys Gly Gly Cys Cys Gly Ala Ala Ala Thr Cys GlyAla 1570 1575 1580 Gly Thr Ala Cys Ala Ala Ala Thr Gly Gly Thr Gly ThrAla Cys Gly 1585 1590 1595 1600 Thr Cys Gly Ala Ala Gly Gly Ala Gly CysCys Gly Ala Ala Thr Thr 1605 1610 1615 Gly Thr Thr Cys Gly Ala Cys AlaGly Cys Gly Gly Thr Cys Ala Ala 1620 1625 1630 Gly Gly Cys Cys Gly AlaCys Gly Cys Gly Ala Ala Cys Ala Thr Cys 1635 1640 1645 Gly Ala Ala CysThr Gly Thr Cys Gly Ala Gly Thr Thr Ala Thr Ala 1650 1655 1660 Ala AlaThr Thr Cys Ala Cys Thr Ala Ala Ala Ala Thr Cys Thr Gly 1665 1670 16751680 Cys Cys Ala Ala Ala Ala Ala Cys Gly Gly Ala Cys Ala Cys Thr Thr1685 1690 1695 Gly Cys Cys Ala Gly Cys Ala Cys Thr Thr Cys Ala Thr CysGly Gly 1700 1705 1710 Gly Gly Ala Cys Cys Thr Ala Cys Thr Cys Thr CysGly Thr Cys Thr 1715 1720 1725 Ala Cys Gly Gly Gly Thr Thr Ala Gly ThrThr Thr Cys Ala Thr Ala 1730 1735 1740 Thr Thr Thr Gly Ala Thr Cys GlyCys Gly Ala Cys Ala Gly Cys Gly 1745 1750 1755 1760 Gly Cys Thr Thr CysThr Ala Cys Thr Thr Thr Cys Thr Thr Cys Ala 1765 1770 1775 Ala Ala ThrAla Thr Thr Thr Thr Thr Cys Cys Cys Thr Gly Cys Cys 1780 1785 1790 AlaGly Cys Cys Thr Cys Gly Thr Cys Gly Thr Ala Gly Thr Thr Thr 1795 18001805 Thr Ala Thr Cys Ala Thr Gly Gly Ala Thr Cys Thr Cys Ala Thr Thr1810 1815 1820 Cys Thr Gly Gly Ala Thr Cys Ala Ala Thr Cys Gly Thr GlyAla Cys 1825 1830 1835 1840 Thr Cys Gly Gly Cys Gly Cys Cys Thr Thr CysGly Cys Gly Ala Ala 1845 1850 1855 Cys Cys Cys Thr Ala Ala Thr Cys GlyGly Thr Ala Cys Gly Ala Thr 1860 1865 1870 Gly Ala Cys Gly Gly Thr GlyCys Thr Cys Ala Cys Thr Gly Ala Gly 1875 1880 1885 Ala Cys Thr Cys AlaThr Cys Thr Thr Ala Thr Gly Ala Cys Cys Gly 1890 1895 1900 Gly Ala AlaCys Cys Ala Ala Thr Cys Gly Ala Cys Gly Thr Cys Thr 1905 1910 1915 1920Thr Cys Cys Ala Cys Cys Ala Gly Thr Thr Gly Cys Cys Thr Ala Thr 19251930 1935 Gly Thr Ala Ala Ala Ala Gly Cys Cys Gly Thr Thr Gly Ala ThrGly 1940 1945 1950 Thr Ala Thr Thr Cys Cys Thr Cys Gly Gly Thr Thr ThrCys Thr Gly 1955 1960 1965 Cys Thr Ala Thr Cys Thr Thr Cys Thr Gly GlyThr Thr Ala Thr Ala 1970 1975 1980 Cys Thr Gly Gly Cys Gly Thr Thr GlyAla Thr Cys Gly Ala Gly Thr 1985 1990 1995 2000 Ala Cys Gly Cys Cys ThrGly Thr Gly Thr Thr Gly Cys Cys Thr Ala 2005 2010 2015 Cys Thr Cys AlaAla Ala Ala Ala Ala Gly Ala Ala Gly Ala Ala Cys 2020 2025 2030 Gly AlaGly Gly Ala Thr Cys Gly Thr Cys Gly Gly Ala Gly Ala Ala 2035 2040 2045Gly Ala Gly Ala Gly Ala Ala Gly Ala Ala Gly Ala Cys Gly Gly Ala 20502055 2060 Gly Cys Ala Thr Ala Ala Ala Cys Cys Thr Gly Cys Thr Cys CysGly 2065 2070 2075 2080 Cys Cys Gly Ala Cys Ala Cys Cys Thr Gly Ala ThrAla Thr Thr Cys 2085 2090 2095 Thr Thr Cys Ala Cys Gly Ala Cys Gly ThrCys Cys Gly Cys Cys Thr 2100 2105 2110 Thr Gly Cys Cys Gly Ala Ala ThrGly Cys Ala Cys Ala Thr Gly Cys 2115 2120 2125 Ala Ala Cys Gly Cys GlyGly Cys Thr Cys Cys Ala Ala Cys Cys Thr 2130 2135 2140 Cys Gly Ala ThrCys Ala Thr Cys Gly Cys Cys Gly Thr Cys Ala Thr 2145 2150 2155 2160 CysAla Ala Gly Cys Ala Gly Thr Cys Gly Ala Ala Thr Cys Gly Ala 2165 21702175 Thr Thr Cys Thr Gly Thr Gly Thr Cys Ala Gly Thr Cys Ala Cys Ala2180 2185 2190 Gly Thr Cys Ala Cys Ala Thr Thr Gly Ala Cys Ala Thr CysGly Thr 2195 2200 2205 Cys Ala Gly Cys Cys Gly Thr Gly Cys Cys Gly CysGly Thr Thr Thr 2210 2215 2220 Cys Cys Thr Cys Thr Thr Gly Thr Thr ThrThr Cys Ala Thr Cys Thr 2225 2230 2235 2240 Thr Gly Thr Thr Cys Ala AlaCys Ala Cys Thr Cys Thr Cys Thr Thr 2245 2250 2255 Cys Thr Gly Gly CysThr Gly Ala Thr Thr Cys Thr Ala Cys Thr Gly 2260 2265 2270 Thr Ala CysAla Ala Ala Thr Cys Cys Ala Ala Gly Cys Gly Thr Cys 2275 2280 2285 ThrGly Cys Cys Gly Thr Ala Thr Ala Thr Thr Ala Gly Thr Gly Ala 2290 22952300 Ala Cys Ala Cys Gly Ala Gly Gly Gly Thr Gly Ala Cys Cys Gly Thr2305 2310 2315 2320 Thr Gly Cys Gly Ala Thr Gly Cys Thr Cys Cys Ala GlyAla Cys Cys 2325 2330 2335 Thr Thr Cys Ala Thr Thr Ala Ala Thr Cys ThrCys Ala Ala Thr Cys 2340 2345 2350 Cys Ala Ala Cys Thr Thr Cys Cys ThrCys Ala Thr Cys Ala Thr Thr 2355 2360 2365 Thr Thr Cys Cys Ala Thr ThrThr Cys Gly Ala Ala Thr Ala Thr Cys 2370 2375 2380 Thr Cys Thr Thr ThrThr Thr Cys Thr Thr Gly Cys Ala Cys Ala Gly 2385 2390 2395 2400 Ala AlaGly Cys Cys Thr Thr Thr Thr Thr Thr Cys Gly Thr Thr Thr 2405 2410 2415Thr Thr Thr Thr Thr Thr Ala Thr Thr Gly Ala Thr Thr Thr Ala Thr 24202425 2430 Thr Thr Thr Thr Ala Cys Gly Gly Ala Thr Thr Thr Thr Thr AlaGly 2435 2440 2445 Ala Thr Ala Ala Thr Gly Cys Ala Cys Ala Gly Ala ThrGly Cys Cys 2450 2455 2460 Thr Cys Ala Thr Thr Gly Cys Thr Cys Ala AlaAla Thr Ala Ala Ala 2465 2470 2475 2480 Thr Thr Thr Ala Thr Thr Thr ThrAla Ala Thr Thr Ala Ala Ala Ala 2485 2490 2495 Ala Ala Ala Ala Ala AlaAla Ala Ala Ala Ala Ala 2500 2505 8 2508 DNA Caenorhabditis elegans 8aagtttgaga gtgatatagg agaaaaacct ccccaacatt ggctcacacc cggattatga 60tcttctgctg ctcctgctgc tccttctgct gtagttgaga cgaagaagaa gaagaagctc 120cattctcgag aaatggctcg tccattcaca cttatcgtac tcctctccgc acatctgtgt 180ctacatgtgg ttgtgacaca ggatgaggac tcacatatca acactcaact cctctcatca 240gttctcgata gactcacgaa tcgcactact tatgataaaa gattacggcc caggtatggt 300gaaaagccag tcgacgttgg aattacgata cacgtttctt caatctctgc agtttcagaa 360gttgatatgg acttcacatt agacttctac atgcgtcaaa cgtggcaaga ccctcgacta 420gccttcggaa gtcttgattt gggactttcc aaagaaatcg actcacttac cgtcggagta 480gactacctgg atagactgtg gaaacccgac acgttcttcc caaatgaaaa gaaatcattc 540ttccacttgg caaccacaca taactcgttc cttcgtatcg agggtgatgg aacggtttat 600actagtcaaa gattaacagt cactgcaacg tgtccaatgg acctgaagct gttcccaatg 660gactctcaac actgtaaact ggaaattgaa agctacgggt acgagacgaa agatatcgac 720tactattggg ggaagaagcg gactgatttg gagataacgg ctgtcaagtt tgataccttc 780cagttgccgc agtttcagcc aacgctgtat tttgtgaata caactaaagc cgagacctca 840tcaggaaaat acgtacgcct ggcgctggaa gtaatattgg ttcgaaatat gggcttctac 900actatgaaca tcgtcatccc atccatcctg atcgtcacca tatcttgggt atcattttgg 960ttgaatcgag aagcttcgcc ggctcgagtt ggattgggtg tgactactgt gctcacaatg 1020acaactctga tcactacaac caataattcg atgccaaaag tgtcttatgt caagggtctg 1080gatgtgtttc ttaatttttg tttcgtaatg gtattcgcct cgttgctcga gtacgccata 1140gtatcctaca tgaataaacg actggtcctc cgacgggaaa aacgaagaaa agccgccgaa 1200caacagcagc gaaacgagat gccaatgttc aacgcgagcc cgaaggccgc caataataat 1260aatccattga tggagatccc agaaaattgt gattgccgga cgattccaat gatgcaacat 1320ccacgtcttg tcacagacgg cgcacatacg ctatggccgg ctccattcgc gcggccgaaa 1380aaggcttcca agacatgctg ccaacgatgg acgcctgcaa aaatcgataa gcttagccga 1440tacggtttcc cattgtcttt ctctatcttc aatatagtct actggttgta tatgaaatat 1500ctaagcttaa actcgtcgga caagatccag gagaacgaca agtggcagca gatccactga 1560tgcgtattcg acggccgaaa tcgagtacaa atggtgtacg tcgaaggagc cgaattgttc 1620gacagcggtc aaggccgacg cgaacatcga actgtcgagt tataaattca ctaaaatctg 1680ccaaaaacgg acacttgcca gcacttcatc ggggacctac tctcgtctac gggttagttt 1740catatttgat cgcgacagcg gcttctactt tcttcaaata tttttccctg ccagcctcgt 1800cgtagtttta tcatggatct cattctggat caatcgtgac tcggcgcctt cgcgaaccct 1860aatcggtacg atgacggtgc tcactgagac tcatcttatg accggaacca atcgacgtct 1920tccaccagtt gcctatgtaa aagccgttga tgtattcctc ggtttctgct atcttctggt 1980tatactggcg ttgatcgagt acgcctgtgt tgcctactca aaaaagaaga acgaggatcg 2040tcggagaaga gagaagaaga cggagcataa acctgctccg ccgacacctg atattcttca 2100cgacgtccgc cttgccgaat gcacatgcaa cgcggctcca acctcgatca tcgccgtcat 2160caagcagtcg aatcgattct gtgtcagtca cagtcacatt gacatcgtca gccgtgccgc 2220gtttcctctt gttttcatct tgttcaacac tctcttctgg ctgattctac tgtacaaatc 2280caagcgtctg ccgtatatta gtgaacacga gggtgaccgt tgcgatgctc cagaccttca 2340ttaatctcaa tccaacttcc tcatcatttt ccatttcgaa tatctctttt tcttgcacag 2400aagccttttt tcgttttttt ttattgattt atttttacgg atttttagat aatgcacaga 2460tgcctcattg ctcaaataaa tttattttaa ttaaaaaaaa aaaaaaaa 2508 9 2601 PRTCaenorhabditis elegans 9 Ala Ala Gly Thr Thr Thr Gly Ala Gly Ala Gly ThrGly Ala Thr Ala 1 5 10 15 Thr Ala Gly Gly Ala Gly Ala Ala Ala Ala AlaCys Cys Thr Cys Cys 20 25 30 Cys Cys Ala Ala Cys Ala Thr Thr Gly Gly CysThr Cys Ala Cys Ala 35 40 45 Cys Cys Cys Gly Gly Ala Thr Thr Ala Thr GlyAla Thr Cys Thr Thr 50 55 60 Cys Thr Gly Cys Thr Gly Cys Thr Cys Cys ThrGly Cys Thr Gly Cys 65 70 75 80 Thr Cys Cys Thr Thr Cys Thr Gly Cys ThrGly Thr Ala Gly Thr Thr 85 90 95 Gly Ala Gly Ala Cys Gly Ala Ala Gly AlaAla Gly Ala Ala Gly Ala 100 105 110 Ala Gly Ala Ala Gly Cys Thr Cys CysAla Thr Thr Cys Thr Cys Gly 115 120 125 Ala Gly Ala Ala Ala Thr Gly GlyCys Thr Cys Gly Thr Cys Cys Ala 130 135 140 Thr Thr Cys Ala Cys Ala CysThr Thr Ala Thr Cys Gly Thr Ala Cys 145 150 155 160 Thr Cys Cys Thr CysThr Cys Cys Gly Cys Ala Cys Ala Thr Cys Thr 165 170 175 Gly Thr Gly ThrCys Thr Ala Cys Ala Thr Gly Thr Gly Gly Thr Thr 180 185 190 Gly Thr GlyAla Cys Ala Cys Ala Gly Gly Ala Thr Gly Ala Gly Gly 195 200 205 Ala CysThr Cys Ala Cys Ala Thr Ala Thr Cys Ala Ala Cys Ala Cys 210 215 220 ThrCys Ala Ala Cys Thr Cys Cys Thr Cys Thr Cys Ala Thr Cys Ala 225 230 235240 Gly Thr Thr Cys Thr Cys Gly Ala Thr Ala Gly Ala Cys Thr Cys Ala 245250 255 Cys Gly Ala Ala Thr Cys Gly Cys Ala Cys Thr Ala Cys Thr Thr Ala260 265 270 Thr Gly Ala Thr Ala Ala Ala Ala Gly Ala Thr Thr Ala Cys GlyGly 275 280 285 Cys Cys Cys Ala Gly Gly Thr Ala Thr Gly Gly Thr Gly AlaAla Ala 290 295 300 Ala Gly Cys Cys Ala Gly Thr Cys Gly Ala Cys Gly ThrThr Gly Gly 305 310 315 320 Ala Ala Thr Thr Ala Cys Gly Ala Thr Ala CysAla Cys Gly Thr Thr 325 330 335 Thr Cys Thr Thr Cys Ala Ala Thr Cys ThrCys Thr Gly Cys Ala Gly 340 345 350 Thr Thr Thr Cys Ala Gly Ala Ala GlyThr Thr Gly Ala Thr Ala Thr 355 360 365 Gly Gly Ala Cys Thr Thr Cys AlaCys Ala Thr Thr Ala Gly Ala Cys 370 375 380 Thr Thr Cys Thr Ala Cys AlaThr Gly Cys Gly Thr Cys Ala Ala Ala 385 390 395 400 Cys Gly Thr Gly GlyCys Ala Ala Gly Ala Cys Cys Cys Thr Cys Gly 405 410 415 Ala Cys Thr AlaGly Cys Cys Thr Thr Cys Gly Gly Ala Ala Gly Thr 420 425 430 Cys Thr ThrGly Ala Thr Thr Thr Gly Gly Gly Ala Cys Thr Thr Thr 435 440 445 Cys CysAla Ala Ala Gly Ala Ala Ala Thr Cys Gly Ala Cys Thr Cys 450 455 460 AlaCys Thr Thr Ala Cys Cys Gly Thr Cys Gly Gly Ala Gly Thr Ala 465 470 475480 Gly Ala Cys Thr Ala Cys Cys Thr Gly Gly Ala Thr Ala Gly Ala Cys 485490 495 Thr Gly Thr Gly Gly Ala Ala Ala Cys Cys Cys Gly Ala Cys Ala Cys500 505 510 Gly Thr Thr Cys Thr Thr Cys Cys Cys Ala Ala Ala Thr Gly AlaAla 515 520 525 Ala Ala Gly Ala Ala Ala Thr Cys Ala Thr Thr Cys Thr ThrCys Cys 530 535 540 Ala Cys Thr Thr Gly Gly Cys Ala Ala Cys Cys Ala CysAla Cys Ala 545 550 555 560 Thr Ala Ala Cys Thr Cys Gly Thr Thr Cys CysThr Thr Cys Gly Thr 565 570 575 Ala Thr Cys Gly Ala Gly Gly Gly Thr GlyAla Thr Gly Gly Ala Ala 580 585 590 Cys Gly Gly Thr Thr Thr Ala Thr AlaCys Thr Ala Gly Thr Cys Ala 595 600 605 Ala Ala Gly Ala Thr Thr Ala AlaCys Ala Gly Thr Cys Ala Cys Thr 610 615 620 Gly Cys Ala Ala Cys Gly ThrGly Thr Cys Cys Ala Ala Thr Gly Gly 625 630 635 640 Ala Cys Cys Thr GlyAla Ala Gly Cys Thr Gly Thr Thr Cys Cys Cys 645 650 655 Ala Ala Thr GlyGly Ala Cys Thr Cys Thr Cys Ala Ala Cys Ala Cys 660 665 670 Thr Gly ThrAla Ala Ala Cys Thr Gly Gly Ala Ala Ala Thr Thr Gly 675 680 685 Ala AlaAla Gly Cys Thr Ala Cys Gly Gly Gly Thr Ala Cys Gly Ala 690 695 700 GlyAla Cys Gly Ala Ala Ala Gly Ala Thr Ala Thr Cys Gly Ala Cys 705 710 715720 Thr Ala Cys Thr Ala Thr Thr Gly Gly Gly Gly Gly Ala Ala Gly Ala 725730 735 Ala Gly Cys Gly Gly Ala Cys Thr Gly Ala Thr Thr Thr Gly Gly Ala740 745 750 Gly Ala Thr Ala Ala Cys Gly Gly Cys Thr Gly Thr Cys Ala AlaGly 755 760 765 Thr Thr Thr Gly Ala Thr Ala Cys Cys Thr Thr Cys Cys AlaGly Thr 770 775 780 Thr Gly Cys Cys Gly Cys Ala Gly Thr Thr Thr Cys AlaGly Cys Cys 785 790 795 800 Ala Ala Cys Gly Cys Thr Gly Thr Ala Thr ThrThr Thr Gly Thr Gly 805 810 815 Ala Ala Thr Ala Cys Ala Ala Cys Thr AlaAla Ala Gly Cys Cys Gly 820 825 830 Ala Gly Ala Cys Cys Thr Cys Ala ThrCys Ala Gly Gly Ala Ala Ala 835 840 845 Ala Thr Ala Cys Gly Thr Ala CysGly Cys Cys Thr Gly Gly Cys Gly 850 855 860 Cys Thr Gly Gly Ala Ala GlyThr Ala Ala Thr Ala Thr Thr Gly Gly 865 870 875 880 Thr Thr Cys Gly AlaAla Ala Thr Ala Thr Gly Gly Gly Cys Thr Thr 885 890 895 Cys Thr Ala CysAla Cys Thr Ala Thr Gly Ala Ala Cys Ala Thr Cys 900 905 910 Gly Thr CysAla Thr Cys Cys Cys Ala Thr Cys Cys Ala Thr Cys Cys 915 920 925 Thr GlyAla Thr Cys Gly Thr Cys Ala Cys Cys Ala Thr Ala Thr Cys 930 935 940 ThrThr Gly Gly Gly Thr Ala Thr Cys Ala Thr Thr Thr Thr Gly Gly 945 950 955960 Thr Thr Gly Ala Ala Thr Cys Gly Ala Gly Ala Ala Gly Cys Thr Thr 965970 975 Cys Gly Cys Cys Gly Gly Cys Thr Cys Gly Ala Gly Thr Thr Gly Gly980 985 990 Ala Thr Thr Gly Gly Gly Thr Gly Thr Gly Ala Cys Thr Ala CysThr 995 1000 1005 Gly Thr Gly Cys Thr Cys Ala Cys Ala Ala Thr Gly AlaCys Ala Ala 1010 1015 1020 Cys Thr Cys Thr Gly Ala Thr Cys Ala Cys ThrAla Cys Ala Ala Cys 1025 1030 1035 1040 Cys Ala Ala Thr Ala Ala Thr ThrCys Gly Ala Thr Gly Cys Cys Ala 1045 1050 1055 Ala Ala Ala Gly Thr GlyThr Cys Thr Thr Ala Thr Gly Thr Cys Ala 1060 1065 1070 Ala Gly Gly GlyThr Cys Thr Gly Gly Ala Thr Gly Thr Gly Thr Thr 1075 1080 1085 Thr CysThr Thr Ala Ala Thr Thr Thr Thr Thr Gly Thr Thr Thr Cys 1090 1095 1100Gly Thr Ala Ala Thr Gly Gly Thr Ala Thr Thr Cys Gly Cys Cys Thr 11051110 1115 1120 Cys Gly Thr Thr Gly Cys Thr Cys Gly Ala Gly Thr Ala CysGly Cys 1125 1130 1135 Cys Ala Thr Ala Gly Thr Ala Thr Cys Cys Thr AlaCys Ala Thr Gly 1140 1145 1150 Ala Ala Thr Ala Ala Ala Cys Gly Ala CysThr Gly Gly Thr Cys Cys 1155 1160 1165 Thr Cys Cys Gly Ala Cys Gly GlyGly Ala Ala Ala Ala Ala Cys Gly 1170 1175 1180 Ala Ala Gly Ala Ala AlaAla Gly Cys Cys Gly Cys Cys Gly Ala Ala 1185 1190 1195 1200 Cys Ala AlaCys Ala Gly Cys Ala Gly Cys Gly Ala Ala Ala Cys Gly 1205 1210 1215 AlaGly Ala Thr Gly Cys Cys Ala Ala Thr Gly Thr Thr Cys Ala Ala 1220 12251230 Cys Gly Cys Gly Ala Gly Cys Cys Cys Gly Ala Ala Gly Gly Cys Cys1235 1240 1245 Gly Cys Cys Ala Ala Thr Ala Ala Thr Ala Ala Thr Thr CysAla Thr 1250 1255 1260 Ala Cys Gly Ala Ala Ala Thr Gly Ala Cys Ala CysThr Thr Ala Thr 1265 1270 1275 1280 Gly Thr Cys Gly Cys Ala Ala Ala AlaThr Thr Cys Gly Ala Cys Gly 1285 1290 1295 Cys Cys Thr Gly Cys Cys AlaAla Ala Ala Gly Cys Thr Ala Thr Gly 1300 1305 1310 Thr Ala Cys Ala GlyGly Cys Thr Gly Ala Cys Thr Thr Gly Thr Ala 1315 1320 1325 Cys Thr ThrThr Gly Cys Cys Gly Gly Ala Cys Ala Cys Ala Ala Thr 1330 1335 1340 ThrCys Cys Thr Cys Thr Ala Thr Gly Ala Ala Thr Cys Cys Ala Thr 1345 13501355 1360 Thr Gly Ala Thr Gly Gly Ala Gly Ala Thr Cys Cys Cys Ala GlyAla 1365 1370 1375 Ala Ala Ala Thr Thr Gly Thr Gly Ala Thr Thr Gly CysCys Gly Gly 1380 1385 1390 Ala Cys Gly Ala Thr Thr Cys Cys Ala Ala ThrGly Ala Thr Gly Cys 1395 1400 1405 Ala Ala Cys Ala Thr Cys Cys Ala CysGly Thr Cys Thr Thr Gly Thr 1410 1415 1420 Cys Ala Cys Ala Gly Ala CysGly Gly Cys Gly Cys Ala Cys Ala Thr 1425 1430 1435 1440 Ala Cys Gly CysThr Ala Thr Gly Gly Cys Cys Gly Gly Cys Thr Cys 1445 1450 1455 Cys AlaThr Thr Cys Gly Cys Gly Cys Gly Gly Cys Cys Gly Ala Ala 1460 1465 1470Ala Ala Ala Gly Gly Cys Thr Thr Cys Cys Ala Ala Gly Ala Cys Ala 14751480 1485 Thr Gly Cys Thr Gly Cys Cys Ala Ala Cys Gly Ala Thr Gly GlyAla 1490 1495 1500 Cys Gly Cys Cys Thr Gly Cys Ala Ala Ala Ala Ala ThrCys Gly Ala 1505 1510 1515 1520 Thr Ala Ala Gly Cys Thr Thr Ala Gly CysCys Gly Ala Thr Ala Cys 1525 1530 1535 Gly Gly Thr Thr Thr Cys Cys CysAla Thr Thr Gly Thr Cys Thr Thr 1540 1545 1550 Thr Cys Thr Cys Thr AlaThr Cys Thr Thr Cys Ala Ala Thr Ala Thr 1555 1560 1565 Ala Gly Thr CysThr Ala Cys Thr Gly Gly Thr Thr Gly Thr Ala Thr 1570 1575 1580 Ala ThrGly Ala Ala Ala Thr Ala Thr Cys Thr Ala Ala Gly Cys Thr 1585 1590 15951600 Thr Ala Ala Ala Cys Thr Cys Gly Thr Cys Gly Gly Ala Cys Ala Ala1605 1610 1615 Gly Ala Thr Cys Cys Ala Gly Gly Ala Gly Ala Ala Cys GlyAla Cys 1620 1625 1630 Ala Ala Gly Thr Gly Gly Cys Ala Gly Cys Ala GlyAla Thr Cys Cys 1635 1640 1645 Ala Cys Thr Gly Ala Thr Gly Cys Gly ThrAla Thr Thr Cys Gly Ala 1650 1655 1660 Cys Gly Gly Cys Cys Gly Ala AlaAla Thr Cys Gly Ala Gly Thr Ala 1665 1670 1675 1680 Cys Ala Ala Ala ThrGly Gly Thr Gly Thr Ala Cys Gly Thr Cys Gly 1685 1690 1695 Ala Ala GlyGly Ala Gly Cys Cys Gly Ala Ala Thr Thr Gly Thr Thr 1700 1705 1710 CysGly Ala Cys Ala Gly Cys Gly Gly Thr Cys Ala Ala Gly Gly Cys 1715 17201725 Cys Gly Ala Cys Gly Cys Gly Ala Ala Cys Ala Thr Cys Gly Ala Ala1730 1735 1740 Cys Thr Gly Thr Cys Gly Ala Gly Thr Thr Ala Thr Ala AlaAla Thr 1745 1750 1755 1760 Thr Cys Ala Cys Thr Ala Ala Ala Ala Thr CysThr Gly Cys Cys Ala 1765 1770 1775 Ala Ala Ala Ala Cys Gly Gly Ala CysAla Cys Thr Thr Gly Cys Cys 1780 1785 1790 Ala Gly Cys Ala Cys Thr ThrCys Ala Thr Cys Gly Gly Gly Gly Ala 1795 1800 1805 Cys Cys Thr Ala CysThr Cys Thr Cys Gly Thr Cys Thr Ala Cys Gly 1810 1815 1820 Gly Gly ThrThr Ala Gly Thr Thr Thr Cys Ala Thr Ala Thr Thr Thr 1825 1830 1835 1840Gly Ala Thr Cys Gly Cys Gly Ala Cys Ala Gly Cys Gly Gly Cys Thr 18451850 1855 Thr Cys Thr Ala Cys Thr Thr Thr Cys Thr Thr Cys Ala Ala AlaThr 1860 1865 1870 Ala Thr Thr Thr Thr Thr Cys Cys Cys Thr Gly Cys CysAla Gly Cys 1875 1880 1885 Cys Thr Cys Gly Thr Cys Gly Thr Ala Gly ThrThr Thr Thr Ala Thr 1890 1895 1900 Cys Ala Thr Gly Gly Ala Thr Cys ThrCys Ala Thr Thr Cys Thr Gly 1905 1910 1915 1920 Gly Ala Thr Cys Ala AlaThr Cys Gly Thr Gly Ala Cys Thr Cys Gly 1925 1930 1935 Gly Cys Gly CysCys Thr Thr Cys Gly Cys Gly Ala Ala Cys Cys Cys 1940 1945 1950 Thr AlaAla Thr Cys Gly Gly Thr Ala Cys Gly Ala Thr Gly Ala Cys 1955 1960 1965Gly Gly Thr Gly Cys Thr Cys Ala Cys Thr Gly Ala Gly Ala Cys Thr 19701975 1980 Cys Ala Thr Cys Thr Thr Ala Thr Gly Ala Cys Cys Gly Gly AlaAla 1985 1990 1995 2000 Cys Cys Ala Ala Thr Cys Gly Ala Cys Gly Thr CysThr Thr Cys Cys 2005 2010 2015 Ala Cys Cys Ala Gly Thr Thr Gly Cys CysThr Ala Thr Gly Thr Ala 2020 2025 2030 Ala Ala Ala Gly Cys Cys Gly ThrThr Gly Ala Thr Gly Thr Ala Thr 2035 2040 2045 Thr Cys Cys Thr Cys GlyGly Thr Thr Thr Cys Thr Gly Cys Thr Ala 2050 2055 2060 Thr Cys Thr ThrCys Thr Gly Gly Thr Thr Ala Thr Ala Cys Thr Gly 2065 2070 2075 2080 GlyCys Gly Thr Thr Gly Ala Thr Cys Gly Ala Gly Thr Ala Cys Gly 2085 20902095 Cys Cys Thr Gly Thr Gly Thr Thr Gly Cys Cys Thr Ala Cys Thr Cys2100 2105 2110 Ala Ala Ala Ala Ala Ala Gly Ala Ala Gly Ala Ala Cys GlyAla Gly 2115 2120 2125 Gly Ala Thr Cys Gly Thr Cys Gly Gly Ala Gly AlaAla Gly Ala Gly 2130 2135 2140 Ala Gly Ala Ala Gly Ala Ala Gly Ala CysGly Gly Ala Gly Cys Ala 2145 2150 2155 2160 Thr Ala Ala Ala Cys Cys ThrGly Cys Thr Cys Cys Gly Cys Cys Gly 2165 2170 2175 Ala Cys Ala Cys CysThr Gly Ala Thr Ala Thr Thr Cys Thr Thr Cys 2180 2185 2190 Ala Cys GlyAla Cys Gly Thr Cys Cys Gly Cys Cys Thr Thr Gly Cys 2195 2200 2205 CysGly Ala Ala Thr Gly Cys Ala Cys Ala Thr Gly Cys Ala Ala Cys 2210 22152220 Gly Cys Gly Gly Cys Thr Cys Cys Ala Ala Cys Cys Thr Cys Gly Ala2225 2230 2235 2240 Thr Cys Ala Thr Cys Gly Cys Cys Gly Thr Cys Ala ThrCys Ala Ala 2245 2250 2255 Gly Cys Ala Gly Thr Cys Gly Ala Ala Thr CysGly Ala Thr Thr Cys 2260 2265 2270 Thr Gly Thr Gly Thr Cys Ala Gly ThrCys Ala Cys Ala Gly Thr Cys 2275 2280 2285 Ala Cys Ala Thr Thr Gly AlaCys Ala Thr Cys Gly Thr Cys Ala Gly 2290 2295 2300 Cys Cys Gly Thr GlyCys Cys Gly Cys Gly Thr Thr Thr Cys Cys Thr 2305 2310 2315 2320 Cys ThrThr Gly Thr Thr Thr Thr Cys Ala Thr Cys Thr Thr Gly Thr 2325 2330 2335Thr Cys Ala Ala Cys Ala Cys Thr Cys Thr Cys Thr Thr Cys Thr Gly 23402345 2350 Gly Cys Thr Gly Ala Thr Thr Cys Thr Ala Cys Thr Gly Thr AlaCys 2355 2360 2365 Ala Ala Ala Thr Cys Cys Ala Ala Gly Cys Gly Thr CysThr Gly Cys 2370 2375 2380 Cys Gly Thr Ala Thr Ala Thr Thr Ala Gly ThrGly Ala Ala Cys Ala 2385 2390 2395 2400 Cys Gly Ala Gly Gly Gly Thr GlyAla Cys Cys Gly Thr Thr Gly Cys 2405 2410 2415 Gly Ala Thr Gly Cys ThrCys Cys Ala Gly Ala Cys Cys Thr Thr Cys 2420 2425 2430 Ala Thr Thr AlaAla Thr Cys Thr Cys Ala Ala Thr Cys Cys Ala Ala 2435 2440 2445 Cys ThrThr Cys Cys Thr Cys Ala Thr Cys Ala Thr Thr Thr Thr Cys 2450 2455 2460Cys Ala Thr Thr Thr Cys Gly Ala Ala Thr Ala Thr Cys Thr Cys Thr 24652470 2475 2480 Thr Thr Thr Thr Cys Thr Thr Gly Cys Ala Cys Ala Gly AlaAla Gly 2485 2490 2495 Cys Cys Thr Thr Thr Thr Thr Thr Cys Gly Thr ThrThr Thr Thr Thr 2500 2505 2510 Thr Thr Thr Ala Thr Thr Gly Ala Thr ThrThr Ala Thr Thr Thr Thr 2515 2520 2525 Thr Ala Cys Gly Gly Ala Thr ThrThr Thr Thr Ala Gly Ala Thr Ala 2530 2535 2540 Ala Thr Gly Cys Ala CysAla Gly Ala Thr Gly Cys Cys Thr Cys Ala 2545 2550 2555 2560 Thr Thr GlyCys Thr Cys Ala Ala Ala Thr Ala Ala Ala Thr Thr Thr 2565 2570 2575 AlaThr Thr Thr Thr Ala Ala Thr Thr Ala Ala Ala Ala Ala Ala Ala 2580 25852590 Ala Ala Ala Ala Ala Ala Ala Ala Ala 2595 2600 10 2601 DNACaenorhabditis elegans 10 aagtttgaga gtgatatagg agaaaaacct ccccaacattggctcacacc cggattatga 60 tcttctgctg ctcctgctgc tccttctgct gtagttgagacgaagaagaa gaagaagctc 120 cattctcgag aaatggctcg tccattcaca cttatcgtactcctctccgc acatctgtgt 180 ctacatgtgg ttgtgacaca ggatgaggac tcacatatcaacactcaact cctctcatca 240 gttctcgata gactcacgaa tcgcactact tatgataaaagattacggcc caggtatggt 300 gaaaagccag tcgacgttgg aattacgata cacgtttcttcaatctctgc agtttcagaa 360 gttgatatgg acttcacatt agacttctac atgcgtcaaacgtggcaaga ccctcgacta 420 gccttcggaa gtcttgattt gggactttcc aaagaaatcgactcacttac cgtcggagta 480 gactacctgg atagactgtg gaaacccgac acgttcttcccaaatgaaaa gaaatcattc 540 ttccacttgg caaccacaca taactcgttc cttcgtatcgagggtgatgg aacggtttat 600 actagtcaaa gattaacagt cactgcaacg tgtccaatggacctgaagct gttcccaatg 660 gactctcaac actgtaaact ggaaattgaa agctacgggtacgagacgaa agatatcgac 720 tactattggg ggaagaagcg gactgatttg gagataacggctgtcaagtt tgataccttc 780 cagttgccgc agtttcagcc aacgctgtat tttgtgaatacaactaaagc cgagacctca 840 tcaggaaaat acgtacgcct ggcgctggaa gtaatattggttcgaaatat gggcttctac 900 actatgaaca tcgtcatccc atccatcctg atcgtcaccatatcttgggt atcattttgg 960 ttgaatcgag aagcttcgcc ggctcgagtt ggattgggtgtgactactgt gctcacaatg 1020 acaactctga tcactacaac caataattcg atgccaaaagtgtcttatgt caagggtctg 1080 gatgtgtttc ttaatttttg tttcgtaatg gtattcgcctcgttgctcga gtacgccata 1140 gtatcctaca tgaataaacg actggtcctc cgacgggaaaaacgaagaaa agccgccgaa 1200 caacagcagc gaaacgagat gccaatgttc aacgcgagcccgaaggccgc caataataat 1260 tcatacgaaa tgacacttat gtcgcaaaat tcgacgcctgccaaaagcta tgtacaggct 1320 gacttgtact ttgccggaca caattcctct atgaatccattgatggagat cccagaaaat 1380 tgtgattgcc ggacgattcc aatgatgcaa catccacgtcttgtcacaga cggcgcacat 1440 acgctatggc cggctccatt cgcgcggccg aaaaaggcttccaagacatg ctgccaacga 1500 tggacgcctg caaaaatcga taagcttagc cgatacggtttcccattgtc tttctctatc 1560 ttcaatatag tctactggtt gtatatgaaa tatctaagcttaaactcgtc ggacaagatc 1620 caggagaacg acaagtggca gcagatccac tgatgcgtattcgacggccg aaatcgagta 1680 caaatggtgt acgtcgaagg agccgaattg ttcgacagcggtcaaggccg acgcgaacat 1740 cgaactgtcg agttataaat tcactaaaat ctgccaaaaacggacacttg ccagcacttc 1800 atcggggacc tactctcgtc tacgggttag tttcatatttgatcgcgaca gcggcttcta 1860 ctttcttcaa atatttttcc ctgccagcct cgtcgtagttttatcatgga tctcattctg 1920 gatcaatcgt gactcggcgc cttcgcgaac cctaatcggtacgatgacgg tgctcactga 1980 gactcatctt atgaccggaa ccaatcgacg tcttccaccagttgcctatg taaaagccgt 2040 tgatgtattc ctcggtttct gctatcttct ggttatactggcgttgatcg agtacgcctg 2100 tgttgcctac tcaaaaaaga agaacgagga tcgtcggagaagagagaaga agacggagca 2160 taaacctgct ccgccgacac ctgatattct tcacgacgtccgccttgccg aatgcacatg 2220 caacgcggct ccaacctcga tcatcgccgt catcaagcagtcgaatcgat tctgtgtcag 2280 tcacagtcac attgacatcg tcagccgtgc cgcgtttcctcttgttttca tcttgttcaa 2340 cactctcttc tggctgattc tactgtacaa atccaagcgtctgccgtata ttagtgaaca 2400 cgagggtgac cgttgcgatg ctccagacct tcattaatctcaatccaact tcctcatcat 2460 tttccatttc gaatatctct ttttcttgca cagaagccttttttcgtttt tttttattga 2520 tttattttta cggattttta gataatgcac agatgcctcattgctcaaat aaatttattt 2580 taattaaaaa aaaaaaaaaa a 2601 11 1128 PRTCaenorhabditis elegans 11 Cys Gly Thr Thr Thr Thr Thr Thr Thr Thr ThrThr Thr Thr Thr Thr 1 5 10 15 Thr Gly Cys Ala Thr Thr Thr Thr Thr CysAla Ala Ala Ala Ala Ala 20 25 30 Ala Ala Ala Ala Ala Ala Ala Ala Thr ThrAla Ala Gly Cys Thr Ala 35 40 45 Gly Thr Ala Gly Thr Cys Thr Gly Thr AlaAla Thr Thr Cys Thr Gly 50 55 60 Thr Thr Gly Cys Thr Thr Thr Thr Cys ThrThr Thr Cys Thr Cys Gly 65 70 75 80 Thr Cys Gly Thr Cys Gly Thr Thr GlyCys Thr Thr Cys Cys Thr Cys 85 90 95 Ala Thr Cys Ala Ala Cys Ala Thr AlaGly Ala Thr Ala Gly Ala Thr 100 105 110 Thr Cys Ala Ala Cys Cys Cys GlyCys Thr Thr Ala Ala Cys Cys Thr 115 120 125 Gly Thr Gly Thr Cys Ala ThrCys Ala Thr Ala Thr Cys Cys Ala Ala 130 135 140 Cys Cys Cys Ala Cys CysAla Ala Cys Cys Ala Thr Gly Ala Ala Gly 145 150 155 160 Cys Cys Cys AlaAla Thr Gly Thr Gly Thr Thr Cys Ala Gly Ala Thr 165 170 175 Gly Cys GlyThr Ala Thr Thr Cys Gly Ala Cys Gly Gly Cys Cys Gly 180 185 190 Ala AlaAla Thr Cys Gly Ala Gly Thr Ala Cys Ala Ala Ala Thr Gly 195 200 205 GlyThr Gly Thr Ala Cys Gly Thr Cys Gly Ala Ala Gly Gly Ala Gly 210 215 220Cys Cys Gly Ala Ala Thr Thr Gly Thr Thr Cys Gly Ala Cys Ala Gly 225 230235 240 Cys Gly Gly Thr Cys Ala Ala Gly Gly Cys Cys Gly Ala Cys Gly Cys245 250 255 Gly Ala Ala Cys Ala Thr Cys Gly Ala Ala Cys Thr Gly Thr CysGly 260 265 270 Ala Gly Thr Thr Ala Thr Ala Ala Ala Thr Thr Cys Ala CysThr Ala 275 280 285 Ala Ala Ala Thr Cys Thr Gly Cys Cys Ala Ala Ala AlaAla Cys Gly 290 295 300 Gly Ala Cys Ala Cys Thr Thr Gly Cys Cys Ala GlyCys Ala Cys Thr 305 310 315 320 Thr Cys Ala Thr Cys Gly Gly Gly Gly AlaCys Cys Thr Ala Cys Thr 325 330 335 Cys Thr Cys Gly Thr Cys Thr Ala CysGly Gly Gly Thr Thr Ala Gly 340 345 350 Thr Thr Thr Cys Ala Thr Ala ThrThr Thr Gly Ala Thr Cys Gly Cys 355 360 365 Gly Ala Cys Ala Gly Cys GlyGly Cys Thr Thr Cys Thr Ala Cys Thr 370 375 380 Thr Thr Cys Thr Thr CysAla Ala Ala Thr Ala Thr Thr Thr Thr Thr 385 390 395 400 Cys Cys Cys ThrGly Cys Cys Ala Gly Cys Cys Thr Cys Gly Thr Cys 405 410 415 Gly Thr AlaGly Thr Thr Thr Thr Ala Thr Cys Ala Thr Gly Gly Ala 420 425 430 Thr CysThr Cys Ala Thr Thr Cys Thr Gly Gly Ala Thr Cys Ala Ala 435 440 445 ThrCys Gly Thr Gly Ala Cys Thr Cys Gly Gly Cys Gly Cys Cys Thr 450 455 460Thr Cys Gly Cys Gly Ala Ala Cys Cys Cys Thr Ala Ala Thr Cys Gly 465 470475 480 Gly Thr Ala Cys Gly Ala Thr Gly Ala Cys Gly Gly Thr Gly Cys Thr485 490 495 Cys Ala Cys Thr Gly Ala Gly Ala Cys Thr Cys Ala Thr Cys ThrThr 500 505 510 Ala Thr Gly Ala Cys Cys Gly Gly Ala Ala Cys Cys Ala AlaThr Cys 515 520 525 Gly Ala Cys Gly Thr Cys Thr Thr Cys Cys Ala Cys CysAla Gly Thr 530 535 540 Thr Gly Cys Cys Thr Ala Thr Gly Thr Ala Ala AlaAla Gly Cys Cys 545 550 555 560 Gly Thr Thr Gly Ala Thr Gly Thr Ala ThrThr Cys Cys Thr Cys Gly 565 570 575 Gly Thr Thr Thr Cys Thr Gly Cys ThrAla Thr Cys Thr Thr Cys Thr 580 585 590 Gly Gly Thr Thr Ala Thr Ala CysThr Gly Gly Cys Gly Thr Thr Gly 595 600 605 Ala Thr Cys Gly Ala Gly ThrAla Cys Gly Cys Cys Thr Gly Thr Gly 610 615 620 Thr Thr Gly Cys Cys ThrAla Cys Thr Cys Ala Ala Ala Ala Ala Ala 625 630 635 640 Gly Ala Ala GlyAla Ala Cys Gly Ala Gly Gly Ala Thr Cys Gly Thr 645 650 655 Cys Gly GlyAla Gly Ala Ala Gly Ala Gly Ala Gly Ala Ala Gly Ala 660 665 670 Ala GlyAla Cys Gly Gly Ala Gly Cys Ala Thr Ala Ala Ala Cys Cys 675 680 685 ThrGly Cys Thr Cys Cys Gly Cys Cys Gly Ala Cys Ala Cys Cys Thr 690 695 700Gly Ala Thr Ala Thr Thr Cys Thr Thr Cys Ala Cys Gly Ala Cys Gly 705 710715 720 Thr Cys Cys Gly Cys Cys Thr Thr Gly Cys Cys Gly Ala Ala Thr Gly725 730 735 Cys Ala Cys Ala Thr Gly Cys Ala Ala Cys Gly Cys Gly Gly CysThr 740 745 750 Cys Cys Ala Ala Cys Cys Thr Cys Gly Ala Thr Cys Ala ThrCys Gly 755 760 765 Cys Cys Gly Thr Cys Ala Thr Cys Ala Ala Gly Cys AlaGly Thr Cys 770 775 780 Gly Ala Ala Thr Cys Gly Ala Thr Thr Cys Thr GlyThr Gly Thr Cys 785 790 795 800 Ala Gly Thr Cys Ala Cys Ala Gly Thr CysAla Cys Ala Thr Thr Gly 805 810 815 Ala Cys Ala Thr Cys Gly Thr Cys AlaGly Cys Cys Gly Thr Gly Cys 820 825 830 Cys Gly Cys Gly Thr Thr Thr CysCys Thr Cys Thr Thr Gly Thr Thr 835 840 845 Thr Thr Cys Ala Thr Cys ThrThr Gly Thr Thr Cys Ala Ala Cys Ala 850 855 860 Cys Thr Cys Thr Cys ThrThr Cys Thr Gly Gly Cys Thr Gly Ala Thr 865 870 875 880 Thr Cys Thr AlaCys Thr Gly Thr Ala Cys Ala Ala Ala Thr Cys Cys 885 890 895 Ala Ala GlyCys Gly Thr Cys Thr Gly Cys Cys Gly Thr Ala Thr Ala 900 905 910 Thr ThrAla Gly Thr Gly Ala Ala Cys Ala Cys Gly Ala Gly Gly Gly 915 920 925 ThrGly Ala Cys Cys Gly Thr Thr Gly Cys Gly Ala Thr Gly Cys Thr 930 935 940Cys Cys Ala Gly Ala Cys Cys Thr Thr Cys Ala Thr Thr Ala Ala Thr 945 950955 960 Cys Thr Cys Ala Ala Thr Cys Cys Ala Ala Cys Thr Thr Cys Cys Thr965 970 975 Cys Ala Thr Cys Ala Thr Thr Thr Thr Cys Cys Ala Thr Thr ThrCys 980 985 990 Gly Ala Ala Thr Ala Thr Cys Thr Cys Thr Thr Thr Thr ThrCys Thr 995 1000 1005 Thr Gly Cys Ala Cys Ala Gly Ala Ala Gly Cys CysThr Thr Thr Thr 1010 1015 1020 Thr Thr Cys Gly Thr Thr Thr Thr Thr ThrThr Thr Thr Ala Thr Thr 1025 1030 1035 1040 Gly Ala Thr Thr Thr Ala ThrThr Thr Thr Thr Ala Cys Gly Gly Ala 1045 1050 1055 Thr Thr Thr Thr ThrAla Gly Ala Thr Ala Ala Thr Gly Cys Ala Cys 1060 1065 1070 Ala Gly AlaThr Gly Cys Cys Thr Cys Ala Thr Thr Gly Cys Thr Cys 1075 1080 1085 AlaAla Ala Thr Ala Ala Ala Thr Thr Thr Ala Thr Thr Thr Thr Ala 1090 10951100 Ala Thr Thr Gly Thr Cys Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala1105 1110 1115 1120 Ala Ala Ala Ala Ala Ala Ala Ala 1125 12 1128 DNACaenorhabditis elegans 12 cgtttttttt tttttttgca tttttcaaaa aaaaaaaaaattaagctagt agtctgtaat 60 tctgttgctt ttctttctcg tcgtcgttgc ttcctcatcaacatagatag attcaacccg 120 cttaacctgt gtcatcatat ccaacccacc aaccatgaagcccaatgtgt tcagatgcgt 180 attcgacggc cgaaatcgag tacaaatggt gtacgtcgaaggagccgaat tgttcgacag 240 cggtcaaggc cgacgcgaac atcgaactgt cgagttataaattcactaaa atctgccaaa 300 aacggacact tgccagcact tcatcgggga cctactctcgtctacgggtt agtttcatat 360 ttgatcgcga cagcggcttc tactttcttc aaatatttttccctgccagc ctcgtcgtag 420 ttttatcatg gatctcattc tggatcaatc gtgactcggcgccttcgcga accctaatcg 480 gtacgatgac ggtgctcact gagactcatc ttatgaccggaaccaatcga cgtcttccac 540 cagttgccta tgtaaaagcc gttgatgtat tcctcggtttctgctatctt ctggttatac 600 tggcgttgat cgagtacgcc tgtgttgcct actcaaaaaagaagaacgag gatcgtcgga 660 gaagagagaa gaagacggag cataaacctg ctccgccgacacctgatatt cttcacgacg 720 tccgccttgc cgaatgcaca tgcaacgcgg ctccaacctcgatcatcgcc gtcatcaagc 780 agtcgaatcg attctgtgtc agtcacagtc acattgacatcgtcagccgt gccgcgtttc 840 ctcttgtttt catcttgttc aacactctct tctggctgattctactgtac aaatccaagc 900 gtctgccgta tattagtgaa cacgagggtg accgttgcgatgctccagac cttcattaat 960 ctcaatccaa cttcctcatc attttccatt tcgaatatctctttttcttg cacagaagcc 1020 ttttttcgtt tttttttatt gatttatttt tacggatttttagataatgc acagatgcct 1080 cattgctcaa ataaatttat tttaattgtc aaaaaaaaaaaaaaaaaa 1128 13 487 PRT Artificial Sequence UNC-49A 13 Met Ala Arg ProPhe Thr Leu Ile Val Leu Leu Ser Ala His Leu Cys 1 5 10 15 Leu His ValVal Val Thr Gln Asp Glu Asp Ser His Ile Asn Thr Gln 20 25 30 Leu Leu SerSer Val Leu Asp Arg Leu Thr Asn Arg Thr Thr Tyr Asp 35 40 45 Lys Arg LeuArg Pro Arg Tyr Gly Glu Lys Pro Val Asp Val Gly Ile 50 55 60 Thr Ile HisVal Ser Ser Ile Ser Ala Val Ser Glu Val Asp Met Asp 65 70 75 80 Phe ThrLeu Asp Phe Tyr Met Arg Gln Thr Trp Gln Asp Pro Arg Leu 85 90 95 Ala PheGly Ser Leu Asp Leu Gly Leu Ser Lys Glu Ile Asp Ser Leu 100 105 110 ThrVal Gly Val Asp Tyr Leu Asp Arg Leu Trp Lys Pro Asp Thr Phe 115 120 125Phe Pro Asn Glu Lys Lys Ser Phe Phe His Leu Ala Thr Thr His Asn 130 135140 Ser Phe Leu Arg Ile Glu Gly Asp Gly Thr Val Tyr Thr Ser Gln Arg 145150 155 160 Leu Thr Val Thr Ala Thr Cys Pro Met Asp Leu Lys Leu Phe ProMet 165 170 175 Asp Ser Gln His Cys Lys Leu Glu Ile Glu Ser Tyr Gly TyrSer Ile 180 185 190 Leu Asp Ile Met Tyr Val Ser His Glu Lys Lys Ser ValSer Thr Glu 195 200 205 Ser Tyr Glu Leu Pro Gln Phe Val Leu Gln Ser IleLys Val Val Asn 210 215 220 His Thr Gln Lys Leu Ser Ser Gly Glu Tyr SerArg Leu Cys Trp Phe 225 230 235 240 Phe Leu Phe Lys Arg Asn Ile Gly PheTyr Ile Ile Gln Ile Tyr Leu 245 250 255 Pro Ser Val Leu Ile Val Val IleSer Trp Val Ser Phe Trp Leu Ser 260 265 270 Arg Asp Ala Thr Pro Ala ArgVal Ala Leu Gly Val Thr Thr Val Leu 275 280 285 Thr Met Thr Thr Leu MetThr Met Thr Asn Ser Ser Met Pro Lys Val 290 295 300 Ser Tyr Val Lys SerIle Asp Ile Phe Leu Gly Val Cys Phe Met Met 305 310 315 320 Val Phe CysSer Leu Leu Glu Tyr Ala Ala Val Gly Tyr Ile Ser Lys 325 330 335 Arg MetLys Leu Val Arg Ala Arg Lys Glu Ser Arg Met Leu Thr Pro 340 345 350 LeuPro His Leu Glu Ser Leu Pro Pro Lys Arg Thr Leu Ser Val Pro 355 360 365Ser Tyr Phe Asn Asn Thr Thr Tyr Arg Pro Phe Tyr Ser Ser Thr Asp 370 375380 Gln Thr Ser Asn Leu Tyr Ile Pro Glu Ser Gln Arg Thr Thr Ile Phe 385390 395 400 Ser Asn Glu Asp Ala Val Pro Asn Glu Leu Thr Pro Met Leu GlyArg 405 410 415 Ser Asn Ser Gln Ala Ser Val Phe Leu Tyr Gln Thr Ala ValIle Ser 420 425 430 Asp Asp Glu Phe Gly Arg Phe Trp Arg Trp Leu Arg ProSer Asn Ile 435 440 445 Asp Lys Tyr Ser Arg Ser Leu Phe Pro Ser Ile PheVal Leu Phe Asn 450 455 460 Val Gly Tyr Trp Ala Tyr Phe Ile Arg Gln SerGln Ile Gln Glu Glu 465 470 475 480 Gln Arg Asn Ser Gln Ile Leu 485 14487 PRT Artificial Sequence UNC-49B.1 14 Met Ala Arg Pro Phe Thr Leu IleVal Leu Leu Ser Ala His Leu Cys 1 5 10 15 Leu His Val Val Val Thr GlnAsp Glu Asp Ser His Ile Asn Thr Gln 20 25 30 Leu Leu Ser Ser Val Leu AspArg Leu Thr Asn Arg Thr Thr Tyr Asp 35 40 45 Lys Arg Leu Arg Pro Arg TyrGly Glu Lys Pro Val Asp Val Gly Ile 50 55 60 Thr Ile His Val Ser Ser IleSer Ala Val Ser Glu Val Asp Met Asp 65 70 75 80 Phe Thr Leu Asp Phe TyrMet Arg Gln Thr Trp Gln Asp Pro Arg Leu 85 90 95 Ala Phe Gly Ser Leu AspLeu Gly Leu Ser Lys Glu Ile Asp Ser Leu 100 105 110 Thr Val Gly Val AspTyr Leu Asp Arg Leu Trp Lys Pro Asp Thr Phe 115 120 125 Phe Pro Asn GluLys Lys Ser Phe Phe His Leu Ala Thr Thr His Asn 130 135 140 Ser Phe LeuArg Ile Glu Gly Asp Gly Thr Val Tyr Thr Ser Gln Arg 145 150 155 160 LeuThr Val Thr Ala Thr Cys Pro Met Asp Leu Lys Leu Phe Pro Met 165 170 175Asp Ser Gln His Cys Lys Leu Glu Ile Glu Ser Tyr Gly Tyr Glu Thr 180 185190 Lys Asp Ile Asp Tyr Tyr Trp Gly Lys Lys Arg Thr Asp Leu Glu Ile 195200 205 Thr Ala Val Lys Phe Asp Thr Phe Gln Leu Pro Gln Phe Gln Pro Thr210 215 220 Leu Tyr Phe Val Asn Thr Thr Lys Ala Glu Thr Ser Ser Gly LysTyr 225 230 235 240 Val Arg Leu Ala Leu Glu Val Ile Leu Val Arg Asn MetGly Phe Tyr 245 250 255 Thr Met Asn Ile Val Ile Pro Ser Ile Leu Ile ValThr Ile Ser Trp 260 265 270 Val Ser Phe Trp Leu Asn Arg Glu Ala Ser ProAla Arg Val Gly Leu 275 280 285 Gly Val Thr Thr Val Leu Thr Met Thr ThrLeu Ile Thr Thr Thr Asn 290 295 300 Asn Ser Met Pro Lys Val Ser Tyr ValLys Gly Leu Asp Val Phe Leu 305 310 315 320 Asn Phe Cys Phe Val Met ValPhe Ala Ser Leu Leu Glu Tyr Ala Ile 325 330 335 Val Ser Tyr Met Asn LysArg Leu Val Leu Arg Arg Glu Lys Arg Arg 340 345 350 Lys Ala Ala Glu GlnGln Gln Arg Asn Glu Met Pro Met Phe Asn Ala 355 360 365 Ser Pro Lys AlaAla Asn Asn Asn Ala Asp Leu Tyr Phe Ala Gly His 370 375 380 Asn Ser SerMet Asn Pro Leu Met Glu Ile Pro Glu Asn Cys Asp Cys 385 390 395 400 ArgThr Ile Pro Met Met Gln His Pro Arg Leu Val Thr Asp Gly Ala 405 410 415His Thr Leu Trp Pro Ala Pro Phe Ala Arg Pro Lys Lys Ala Ser Lys 420 425430 Thr Cys Cys Gln Arg Trp Thr Pro Ala Lys Ile Asp Lys Leu Ser Arg 435440 445 Tyr Gly Phe Pro Leu Ser Phe Ser Ile Phe Asn Ile Val Tyr Trp Leu450 455 460 Tyr Met Lys Tyr Leu Ser Leu Asn Ser Ser Asp Lys Ile Gln GluAsn 465 470 475 480 Asp Lys Trp Gln Gln Ile His 485 15 506 PRTArtificial Sequence UNC-49B.3 15 Met Ala Arg Pro Phe Thr Leu Ile Val LeuLeu Ser Ala His Leu Cys 1 5 10 15 Leu His Val Val Val Thr Gln Asp GluAsp Ser His Ile Asn Thr Gln 20 25 30 Leu Leu Ser Ser Val Leu Asp Arg LeuThr Asn Arg Thr Thr Tyr Asp 35 40 45 Lys Arg Leu Arg Pro Arg Tyr Gly GluLys Pro Val Asp Val Gly Ile 50 55 60 Thr Ile His Val Ser Ser Ile Ser AlaVal Ser Glu Val Asp Met Asp 65 70 75 80 Phe Thr Leu Asp Phe Tyr Met ArgGln Thr Trp Gln Asp Pro Arg Leu 85 90 95 Ala Phe Gly Ser Leu Asp Leu GlyLeu Ser Lys Glu Ile Asp Ser Leu 100 105 110 Thr Val Gly Val Asp Tyr LeuAsp Arg Leu Trp Lys Pro Asp Thr Phe 115 120 125 Phe Pro Asn Glu Lys LysSer Phe Phe His Leu Ala Thr Thr His Asn 130 135 140 Ser Phe Leu Arg IleGlu Gly Asp Gly Thr Val Tyr Thr Ser Gln Arg 145 150 155 160 Leu Thr ValThr Ala Thr Cys Pro Met Asp Leu Lys Leu Phe Pro Met 165 170 175 Asp SerGln His Cys Lys Leu Glu Ile Glu Ser Tyr Gly Tyr Glu Thr 180 185 190 LysAsp Ile Asp Tyr Tyr Trp Gly Lys Lys Arg Thr Asp Leu Glu Ile 195 200 205Thr Ala Val Lys Phe Asp Thr Phe Gln Leu Pro Gln Phe Gln Pro Thr 210 215220 Leu Tyr Phe Val Asn Thr Thr Lys Ala Glu Thr Ser Ser Gly Lys Tyr 225230 235 240 Val Arg Leu Ala Leu Glu Val Ile Leu Val Arg Asn Met Gly PheTyr 245 250 255 Thr Met Asn Ile Val Ile Pro Ser Ile Leu Ile Val Thr IleSer Trp 260 265 270 Val Ser Phe Trp Leu Asn Arg Glu Ala Ser Pro Ala ArgVal Gly Leu 275 280 285 Gly Val Thr Thr Val Leu Thr Met Thr Thr Leu IleThr Thr Thr Asn 290 295 300 Asn Ser Met Pro Lys Val Ser Tyr Val Lys GlyLeu Asp Val Phe Leu 305 310 315 320 Asn Phe Cys Phe Val Met Val Phe AlaSer Leu Leu Glu Tyr Ala Ile 325 330 335 Val Ser Tyr Met Asn Lys Arg LeuVal Leu Arg Arg Glu Lys Arg Arg 340 345 350 Lys Ala Ala Glu Gln Gln GlnArg Asn Glu Met Pro Met Phe Asn Ala 355 360 365 Ser Pro Lys Ala Ala AsnAsn Asn Ser Tyr Glu Met Thr Leu Met Ser 370 375 380 Gln Asn Ser Thr ProAla Lys Ser Tyr Val Gln Ala Asp Leu Tyr Phe 385 390 395 400 Ala Gly HisAsn Ser Ser Met Asn Pro Leu Met Glu Ile Pro Glu Asn 405 410 415 Cys AspCys Arg Thr Ile Pro Met Met Gln His Pro Arg Leu Val Thr 420 425 430 AspGly Ala His Thr Leu Trp Pro Ala Pro Phe Ala Arg Pro Lys Lys 435 440 445Ala Ser Lys Thr Cys Cys Gln Arg Trp Thr Pro Ala Lys Ile Asp Lys 450 455460 Leu Ser Arg Tyr Gly Phe Pro Leu Ser Phe Ser Ile Phe Asn Ile Val 465470 475 480 Tyr Trp Leu Tyr Met Lys Tyr Leu Ser Leu Asn Ser Ser Asp LysIle 485 490 495 Gln Glu Asn Asp Lys Trp Gln Gln Ile His 500 505 16 475PRT Artificial Sequence UNC-49B.2 16 Met Ala Arg Pro Phe Thr Leu Ile ValLeu Leu Ser Ala His Leu Cys 1 5 10 15 Leu His Val Val Val Thr Gln AspGlu Asp Ser His Ile Asn Thr Gln 20 25 30 Leu Leu Ser Ser Val Leu Asp ArgLeu Thr Asn Arg Thr Thr Tyr Asp 35 40 45 Lys Arg Leu Arg Pro Arg Tyr GlyGlu Lys Pro Val Asp Val Gly Ile 50 55 60 Thr Ile His Val Ser Ser Ile SerAla Val Ser Glu Val Asp Met Asp 65 70 75 80 Phe Thr Leu Asp Phe Tyr MetArg Gln Thr Trp Gln Asp Pro Arg Leu 85 90 95 Ala Phe Gly Ser Leu Asp LeuGly Leu Ser Lys Glu Ile Asp Ser Leu 100 105 110 Thr Val Gly Val Asp TyrLeu Asp Arg Leu Trp Lys Pro Asp Thr Phe 115 120 125 Phe Pro Asn Glu LysLys Ser Phe Phe His Leu Ala Thr Thr His Asn 130 135 140 Ser Phe Leu ArgIle Glu Gly Asp Gly Thr Val Tyr Thr Ser Gln Arg 145 150 155 160 Leu ThrVal Thr Ala Thr Cys Pro Met Asp Leu Lys Leu Phe Pro Met 165 170 175 AspSer Gln His Cys Lys Leu Glu Ile Glu Ser Tyr Gly Tyr Glu Thr 180 185 190Lys Asp Ile Asp Tyr Tyr Trp Gly Lys Lys Arg Thr Asp Leu Glu Ile 195 200205 Thr Ala Val Lys Phe Asp Thr Phe Gln Leu Pro Gln Phe Gln Pro Thr 210215 220 Leu Tyr Phe Val Asn Thr Thr Lys Ala Glu Thr Ser Ser Gly Lys Tyr225 230 235 240 Val Arg Leu Ala Leu Glu Val Ile Leu Val Arg Asn Met GlyPhe Tyr 245 250 255 Thr Met Asn Ile Val Ile Pro Ser Ile Leu Ile Val ThrIle Ser Trp 260 265 270 Val Ser Phe Trp Leu Asn Arg Glu Ala Ser Pro AlaArg Val Gly Leu 275 280 285 Gly Val Thr Thr Val Leu Thr Met Thr Thr LeuIle Thr Thr Thr Asn 290 295 300 Asn Ser Met Pro Lys Val Ser Tyr Val LysGly Leu Asp Val Phe Leu 305 310 315 320 Asn Phe Cys Phe Val Met Val PheAla Ser Leu Leu Glu Tyr Ala Ile 325 330 335 Val Ser Tyr Met Asn Lys ArgLeu Val Leu Arg Arg Glu Lys Arg Arg 340 345 350 Lys Ala Ala Glu Gln GlnGln Arg Asn Glu Met Pro Met Phe Asn Ala 355 360 365 Ser Pro Lys Ala AlaAsn Asn Asn Asn Pro Leu Met Glu Ile Pro Glu 370 375 380 Asn Cys Asp CysArg Thr Ile Pro Met Met Gln His Pro Arg Leu Val 385 390 395 400 Thr AspGly Ala His Thr Leu Trp Pro Ala Pro Phe Ala Arg Pro Lys 405 410 415 LysAla Ser Lys Thr Cys Cys Gln Arg Trp Thr Pro Ala Lys Ile Asp 420 425 430Lys Leu Ser Arg Tyr Gly Phe Pro Leu Ser Phe Ser Ile Phe Asn Ile 435 440445 Val Tyr Trp Leu Tyr Met Lys Tyr Leu Ser Leu Asn Ser Ser Asp Lys 450455 460 Ile Gln Glu Asn Asp Lys Trp Gln Gln Ile His 465 470 475 17 448PRT Artificial Sequence UNC-49C 17 Met Ala Arg Pro Phe Thr Leu Ile ValLeu Leu Ser Ala His Leu Cys 1 5 10 15 Leu His Val Val Val Thr Gln AspGlu Asp Ser His Ile Asn Thr Gln 20 25 30 Leu Leu Ser Ser Val Leu Asp ArgLeu Thr Asn Arg Thr Thr Tyr Asp 35 40 45 Lys Arg Leu Arg Pro Arg Tyr GlyGlu Lys Pro Val Asp Val Gly Ile 50 55 60 Thr Ile His Val Ser Ser Ile SerAla Val Ser Glu Val Asp Met Asp 65 70 75 80 Phe Thr Leu Asp Phe Tyr MetArg Gln Thr Trp Gln Asp Pro Arg Leu 85 90 95 Ala Phe Gly Ser Leu Asp LeuGly Leu Ser Lys Glu Ile Asp Ser Leu 100 105 110 Thr Val Gly Val Asp TyrLeu Asp Arg Leu Trp Lys Pro Asp Thr Phe 115 120 125 Phe Pro Asn Glu LysLys Ser Phe Phe His Leu Ala Thr Thr His Asn 130 135 140 Ser Phe Leu ArgIle Glu Gly Asp Gly Thr Val Tyr Thr Ser Gln Arg 145 150 155 160 Leu ThrVal Thr Ala Thr Cys Pro Met Asp Leu Lys Leu Phe Pro Met 165 170 175 AspSer Gln His Cys Lys Leu Glu Ile Glu Ser Tyr Ala Tyr Ser Thr 180 185 190Ala Glu Ile Glu Tyr Lys Trp Cys Thr Ser Lys Glu Pro Asn Cys Ser 195 200205 Thr Ala Val Lys Ala Asp Ala Asn Ile Glu Leu Ser Ser Tyr Lys Phe 210215 220 Thr Lys Ile Cys Gln Lys Arg Thr Leu Ala Ser Thr Ser Ser Gly Thr225 230 235 240 Tyr Ser Arg Leu Arg Val Ser Phe Ile Phe Asp Arg Asp SerGly Phe 245 250 255 Tyr Phe Leu Gln Ile Phe Phe Pro Ala Ser Leu Val ValVal Leu Ser 260 265 270 Trp Ile Ser Phe Trp Ile Asn Arg Asp Ser Ala ProSer Arg Thr Leu 275 280 285 Ile Gly Thr Met Thr Val Leu Thr Glu Thr HisLeu Met Thr Gly Thr 290 295 300 Asn Arg Arg Leu Pro Pro Val Ala Tyr ValLys Ala Val Asp Val Phe 305 310 315 320 Leu Gly Phe Cys Tyr Leu Leu ValIle Leu Ala Leu Ile Glu Tyr Ala 325 330 335 Cys Val Ala Tyr Ser Lys LysLys Asn Glu Asp Arg Arg Arg Arg Glu 340 345 350 Lys Lys Thr Glu His LysPro Ala Pro Pro Thr Pro Asp Ile Leu His 355 360 365 Asp Val Arg Leu AlaGlu Cys Thr Cys Asn Ala Ala Pro Thr Ser Ile 370 375 380 Ile Ala Val IleLys Gln Ser Asn Arg Phe Cys Val Ser His Ser His 385 390 395 400 Ile AspIle Val Ser Arg Ala Ala Phe Pro Leu Val Phe Ile Leu Phe 405 410 415 AsnThr Leu Phe Trp Leu Ile Leu Leu Tyr Lys Ser Lys Arg Leu Pro 420 425 430Tyr Ile Ser Glu His Glu Gly Asp Arg Cys Asp Ala Pro Asp Leu His 435 440445 18 264 PRT Artificial Sequence UNC-49C short 18 Met Cys Ser Asp AlaTyr Ser Thr Ala Glu Ile Glu Tyr Lys Trp Cys 1 5 10 15 Thr Ser Lys GluPro Asn Cys Ser Thr Ala Val Lys Ala Asp Ala Asn 20 25 30 Ile Glu Leu SerSer Tyr Lys Phe Thr Lys Ile Cys Gln Lys Arg Thr 35 40 45 Leu Ala Ser ThrSer Ser Gly Thr Tyr Ser Arg Leu Arg Val Ser Phe 50 55 60 Ile Phe Asp ArgAsp Ser Gly Phe Tyr Phe Leu Gln Ile Phe Phe Pro 65 70 75 80 Ala Ser LeuVal Val Val Leu Ser Trp Ile Ser Phe Trp Ile Asn Arg 85 90 95 Asp Ser AlaPro Ser Arg Thr Leu Ile Gly Thr Met Thr Val Leu Thr 100 105 110 Glu ThrHis Leu Met Thr Gly Thr Asn Arg Arg Leu Pro Pro Val Ala 115 120 125 TyrVal Lys Ala Val Asp Val Phe Leu Gly Phe Cys Tyr Leu Leu Val 130 135 140Ile Leu Ala Leu Ile Glu Tyr Ala Cys Val Ala Tyr Ser Lys Lys Lys 145 150155 160 Asn Glu Asp Arg Arg Arg Arg Glu Lys Lys Thr Glu His Lys Pro Ala165 170 175 Pro Pro Thr Pro Asp Ile Leu His Asp Val Arg Leu Ala Glu CysThr 180 185 190 Cys Asn Ala Ala Pro Thr Ser Ile Ile Ala Val Ile Lys GlnSer Asn 195 200 205 Arg Phe Cys Val Ser His Ser His Ile Asp Ile Val SerArg Ala Ala 210 215 220 Phe Pro Leu Val Phe Ile Leu Phe Asn Thr Leu PheTrp Leu Ile Leu 225 230 235 240 Leu Tyr Lys Ser Lys Arg Leu Pro Tyr IleSer Glu His Glu Gly Asp 245 250 255 Arg Cys Asp Ala Pro Asp Leu His 260

1. A nematode neuromuscular juntion GABA receptor complex of the formulaI A-B  Formula I wherein A an amino acid sequence selected from thegroup consisting of: (a) an amino acid sequence which is encoded by anucleic acid sequence which has at least 80% identity to SEQ ID NO 2;wherein said identity can be determined using the DNAsis computerprogram and default parameters; and (b) an amino acid sequence which hasat least 80% identity to SEQ ID NO 1; wherein said identity can bedetermined using the DNAsis computer program and default parameters; andwherein B is an amino acid sequence selected from the group consistingof: (a) an amino acid sequence which is encoded by a nucleic acidsequence which has at least 80% identity to SEQ ID NO 4; wherein saididentity can be determined using the DNAsis computer program and defaultparameters; and (b) an amino acid sequence which has at least 80%identity to SEQ ID NO 3; wherein said identity can be determined usingthe DNAsis computer program and default parameters; and wherein A-B is aheteropentamer which comprises 1 to 4 A amino acid sequences and 1 to 4B amino acid sequences.
 2. A homopentamer nematode neuromuscular juntionGABA receptor complex of the formula II B₅  Formula II wherein B is anamino acid sequence selected from the group consisting of: (a) an aminoacid sequence which is encoded by a nucleic acid sequence which has atleast 80% identity to SEQ ID NO 4; wherein said to identity can bedetermined using the DNAsis computer program and default parameters; and(b) an amino acid sequence which has at least 80% identity to SEQ ID NO3; wherein said identity can be determined using the DNAsis computerprogram and default parameters.
 3. A nematode neuromuscular junctionGABA receptor complex subunit, comprising an amino acid sequenceselected from the group consisting of: (a) an amino acid sequence whichis encoded by a nucleic acid sequence which has at least 80% identity toSEQ ID NO 2; wherein said identity can be determined using the DNAsiscomputer program and default parameters; and (b) an amino acid sequencewhich has at least 80% identity to SEQ ID NO 1; wherein said identitycan be determined using the DNAsis computer program and defaultparameters.
 4. A nematode neuromuscular junction GABA receptor complexsubunit, comprising an amino acid sequence selected from the groupconsisting of: (a) an amino acid sequence which is encoded by a nucleicacid sequence which has at least 80% identity to SEQ ID NO 4; whereinsaid identity can be determined using the DNAsis computer program anddefault parameters; and (b) an amino acid sequence which has at least80% identity to SEQ ID NO 3; wherein said identity can be determinedusing the DNAsis computer program and default parameters.
 5. A nucleicacid compound which encodes a nematode neuromuscular junction GABAreceptor complex subunit, comprising: (a) a nucleic acid sequence whichhas at least 80% identity to SEQ ID NO 2; wherein said identity can bedetermined using the DNAsis computer program and default parameters; (b)a nucleic acid which encodes an amino acid sequence which has at least80% identity to SEQ ID NO 1; wherein said identity can be determinedusing the DNAsis computer program and default parameters; (c) a nucleicacid sequence which is an allelic variant of SEQ ID NO 2: and (d) anucleic acid sequence fully complementary to a nucleic acid sequenceselected from the group consisting of: a nucleic acid sequence of (a); anucleic acid sequence of (b); and a nucleic acid sequence of (c).
 6. Anucleic acid compound which encodes a nematode neuromuscular junctionGABA receptor complex subunit, comprising a nucleic acid sequenceselected from the group consisting of: (a) a nucleic acid sequence whichhas at least 80% identity to SEQ ID NO 4; wherein said identity can bedetermined using the DNAsis computer program and default parameters; (b)a nucleic acid which encodes an amino acid sequence which has at least80% identity to SEQ ID NO 3; wherein said identity can be determinedusing the DNAsis computer program and default parameters; (c) a nucleicacid sequence which is an allelic variant of SEQ ID NO 4; and (d) anucleic acid sequence fully complementary to a nucleic acid sequenceselected from the group consisting of: a nucleic acid sequence of (a); anucleic acid sequence of (b); and a nucleic acid sequence of (c).
 7. Anucleic acid compound which encodes a nematode neuromuscular junctionGABA receptor complex, comprising a first and second nucleic acidsequence, wherein said first nucleic acid sequence is selected from thegroup consisting of: (a) a nucleic acid sequence which has at least 80%identity to SEQ ID NO 2; wherein said identity can be determined usingthe DNAsis computer program and default parameters; (b) a nucleic acidwhich encodes an amino acid sequence which has at least 80% identity toSEQ ID NO 1; wherein said identity can be determined using the DNAsiscomputer program and default parameters; (c) a nucleic acid sequencewhich is an allelic variant of SEQ ID NO 2; and (d) a nucleic acidsequence which has at least 80% identity to SEQ ID NO 4; wherein saididentity can be determined using the DNAsis computer program and defaultparameters; (e) a nucleic acid which encodes an amino acid sequencewhich has at least 80% identity to SEQ ID NO 3; wherein said identitycan be determined using the DNAsis computer program and defaultparameters; (f) a nucleic acid sequence which is an allelic variant ofSEQ ID NO 4; and wherein said second nucleic acid sequence is selectedfrom the group consisting of: (a) a nucleic acid sequence which has atleast 80% identity to SEQ ID NO 4; wherein said identity can bedetermined using the DNAsis computer program and default parameters; (b)a nucleic acid which encodes an amino acid sequence which has at least80% identity to SEQ ID NO 3; wherein said identity can be determinedusing the DNAsis computer program and default parameters; (c) a nucleicacid sequence which is an allelic variant of SEQ ID NO 4; and.
 8. Anisolated antibody selective for a GABA receptor complex of claim
 1. 9.An isolated antibody selective for a GABA receptor complex of claim 2.10. An isolated antibody selective for a GABA receptor complex subunitof claim
 3. 11. An isolated antibody selective for a GABA receptorcomplex subunit of claim
 4. 12. A method to determine a test substance'sability to interact with a nematode neuromuscular junction GABA receptorcomplex, comprising contacting a receptor complex of claim 1 with a testsubstance and determining whether said test substance and said receptorcomplex interact.
 13. A method to determine a test substance's abilityto interact with a nematode neuromuscular junction GABA receptorcomplex, comprising contacting a receptor complex of claim 2 with a testsubstance and determining whether said test substance and said receptorcomplex interact.
 14. A method to determine a test substance's abilityto interact with a nematode neuromuscular junction GABA receptor complexsubunit, comprising contacting a receptor complex subunit of claim 3with a test substance and determining whether said test substance andsaid receptor complex subunit interact.
 15. A method to determine a testsubstance's ability to interact with a nematode neuromuscular junctionGABA receptor complex subunit, comprising contacting a receptor complexsubunit of claim 4 with a test substance and determining whether saidtest substance and said receptor complex subunit interact.
 16. A methodto detect GABA receptors in a test sample, comprising: (a) immobilizinga test sample on a substrate; (b) contacting the test sample with anantibody of claim 8 under conditions suitable for formation of a GABAreceptor:antibody complex bound to the substrate; (c) removing non-boundmaterial from the substrate under conditions that retain GABA:antibodycomplex binding to the substrate; and (d) detecting the presence of theGABA receptor:antibody complex.
 17. A method to detect GABA receptors ina test sample, comprising: (a) immobilizing a test sample on asubstrate; (b) contacting the test sample with an antibody of claim 9under conditions suitable for formation of a GABA receptor:antibodycomplex bound to the substrate; (c) removing non-bound material from thesubstrate under conditions that retain GABA:antibody complex binding tothe substrate; and (d) detecting the presence of the GABAreceptor:antibody complex.
 18. A method to detect GABA receptors in atest sample, comprising: (a) immobilizing a test sample on a substrate;(b) contacting the test sample with an antibody of claim 10 underconditions suitable for formation of a GABA receptor:antibody complexbound to the substrate; (c) removing non-bound material from thesubstrate under conditions that retain GABA:antibody complex binding tothe substrate; and (d) detecting the presence of the GABAreceptor:antibody complex.
 19. A method to detect GABA receptors in atest sample, comprising: (a) immobilizing a test sample on a substrate;(b) contacting the test sample with an antibody of claim 11 underconditions suitable for formation of a GABA receptor:antibody complexbound to the substrate; (c) removing non-bound material from thesubstrate under conditions that retain GABA:antibody complex binding tothe substrate; and (d) detecting the presence of the GABAreceptor:antibody complex.
 20. An isolated nucleic acid compound ofclaim 5 which comprises a nucleic acid sequence selected from the groupconsisting of: SEQ ID NO 2; SEQ ID NO 4; SEQ ID NO 6; SEQ ID NO 8; SEQID NO 10; and SEQ ID NO
 12. 21. An isolated amino acid compound of claim3 which comprises a nucleic acid sequence selected from the groupconsisting of: SEQ ID NO 1; SEQ ID NO 3, SEQ ID NO 5; SEQ ID NO 7; SEQID NO 9; SEQ ID NO 11.